Nanocomposites and methods of making same

ABSTRACT

A nanoagent includes at least one nanocomposite. The nanocomposite includes at least one gold nanorod, a silver layer coated on an outer surface of the gold nanorod, a Raman reporter molecule layer coated on the silver layer, a pegylated layer coated on the Raman reporter molecule layer, an active layer conjugated to the pegylated layer. the active layer includes at least one of a targeting molecule configured to bind to a target of interest, and a functional molecule configured to interact with the target of interest. The silver layer has silver nanoparticles. The Raman reporter molecule layer has Raman reporter molecules that are detectable by surface enhanced Raman spectroscopy (SERS). The pegylated layer has at least one of thiolated polyethylene glycol (HS-PEG), thiolated polyethylene glycol acid (HS-PEG-COOH) and HS-PEG-NHx.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 14/513,744, filed on Oct. 14, 2014, entitled “NANOCOMPOSITES,METHODS OF MAKING SAME, AND APPLICATIONS OF SAME FOR MULTICOLOR SURFACEENHANCED RAMAN SPECTROSCOPY (SERS) DETECTIONS,” by Alexandru S. Biris,Zeid Nima and Yang Xu, which is incorporated herein by reference in itsentirety and which claims priority to and the benefit of, pursuant to 35U.S.C. §119(e), U.S. provisional patent application Ser. No. 61/891,006,filed on Oct. 15, 2013, entitled “MULTICOLOR SERS DETECTION AND IMAGINGOF CANCER CELLS IN BLOOD USING SILVER DECORATED GOLD NANOROD,” byAlexandru S. Biris, Zeid Nima and Yang Xu, which is incorporated hereinby reference in its entirety.

Some references, which may include patents, patent applications, andvarious publications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entiretiesand to the same extent as if each reference was individuallyincorporated by reference. In terms of notation, hereinafter, “[n]”represents the nth reference cited in the reference list. For example,[3] represents the third reference cited in the reference list, namely,Nima, Z. A. et al. Single-walled carbon nanotubes as specific targetingand Raman spectroscopic agents for detection and discrimination ofsingle human breast cancer cells. Journal of Biomedical Optics 18,055003-055003 (2013).

STATEMENT AS TO RIGHTS UNDER FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under grant numberW81XWH-10-2-0130 awarded by the Department of Defense. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to nanocomposites and methods ofmaking the same.

BACKGROUND OF THE INVENTION

The background description provided herein is for the purpose ofgenerally presenting the context of the present invention. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description that may nototherwise qualify as prior art at the time of filing, are neitherexpressly nor impliedly admitted as prior art against the presentinvention.

Targeting, imaging and treatment of cancer cells using biocompatiblenanomaterials is one ultimate goal for a versatile number of studies indifferent fields of science, engineering, and medicine [1-9].Nanomaterials are widely investigated and tested by researchers fromdifferent fields due to their unique features not observed at themacroscale of the same material [1, 2]. However, there are stillchallenges in the field to discover nanoagents that provide sensitiveand accurate targeting, detection, treatment, and monitoring of cancercells.

Therefore, a heretofore unaddressed need exists in the art to addressthe aforementioned deficiencies and inadequacies.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to a nanoagent for surfaceenhanced Raman spectroscopy (SERS) detection, treatment and monitoringof a target of interest. In certain embodiments, the nanoagent includesat least one nanocomposite.

In one embodiment, the nanocomposite includes at least one gold nanorod,a silver layer coated on an outer surface of the gold nanorod and havingsilver nanoparticles, a Raman reporter molecule layer coated on thesilver layer, a pegylated layer coated on the Raman reporter moleculelayer and having at least one of thiolated polyethylene glycol (HS-PEG),thiolated polyethylene glycol acid (HS-PEG-COOH) and HS-PEG-NHx, and anactive layer conjugated to the pegylated layer. The active layerincludes at least one of a targeting molecule configured to bind to atarget of interest, and a functional molecule configured to interactwith the target of interest.

In certain embodiments, the target of interest is at least one of acancer cell, a pathogen, or a plant, and the functional molecule is avirus, a phage, a drug, a growth factor, antibiotics, a gene, a plasmid,a vaccine, a plant growth agent, an anti-fungal, a fertilizer,herbicides, an antibody that specifically binds to S100 calcium-bindingprotein B (S100B) or other biological active molecules. In oneembodiment, the target of interest is a cancer cell, and the functionalmolecule is a virus that is capable of reproducing in the cancer cell,such that the cancer cell is disrupted by the virus or the virus inducesapoptosis of the cancer cell. In one embodiment, the active layerincludes a biomarker of traumatic brain injury (TBI) or an antibody thatspecifically binds to the biomarker. In one embodiment, the biomarker ofTBI includes at least one of S100B, neuron-specific enolase (NSE),myelin basic protein (MBP) caspase-3, interleukins, tau protein,neurofilament light polypeptide (NEFL), neurofilament heavy polypeptide(NEFH), glial fibrillary acidic protein, amyloid precursor protein(APP), and amyloid.

In one embodiment, the gold nanorod has an aspect ratio (AR) in therange of about 1-9. In one embodiment, the gold nanorod has the AR in arange of about 2-5. In one embodiment, the gold nanorod has the AR in arange of about 2.77-3.23.

In one embodiment, the gold nanorod has a length in the range of about10-100 nm and has a diameter in the range of about 1-40 nm,respectively. In one embodiment, the gold nanorod has the length in therange of about 35.20-36.80 nm and has the diameter in the range of about11.59-12.41 nm, respectively.

In one embodiment, the silver layer has a thickness in a range of about0.5-5 nm. In one embodiment, the silver layer has the thickness of about1-2 nm. In one embodiment, the silver layer has the thickness of about1.7 nm.

In one embodiment, the Raman reporter molecule layer includes4-mercaptobenzoic acid (4MBA), p-aminothiophenol (PATP),p-nitrothiophenol (PNTP), 4-(methylsulfanyl)thiophenol (4MSTP), or othermolecules with unique Raman spectra and intense Raman peak intensities.

In one embodiment, the HS-PEG has a molecular weight in a range of about1.5-15 kilo Dalton (kD) and the HS-PEG-COOH has a molecular weight in arange of about 1-10 kD. In one embodiment, the HS-PEG has the molecularweight of about 5 kD and the HS-PEG-COOH has the molecular weight ofabout 3 kD.

In one embodiment, the molecules of the antibody are conjugated to thecorresponding pegylated layer through the carboxylic group of theHS-PEG-COOH or the amine group of HS-PEG-NHx.

In one embodiment, the targeting molecule is an antibody, and theantibody includes anti-epithelial cell adhesion molecule antibody(anti-EpCAM), anti-CD44 antibody, anti-insulin-like growth factor 1receptor antibody (anti-IGF-1), anti-Keratin 18 antibody, or one or moreantibodies specific to the target of interest.

In one embodiment, the at least one nanocomposite includes a firstnanocomposite, a second nanocomposite, a third nanocomposite, and afourth nanocomposite. The Raman reporter molecule layer of the firstnanocomposite includes 4-mercaptobenzoic acid (4MBA), and the antibodyof the first nanocomposite is anti-epithelial cell adhesion moleculeantibody (anti-EpCAM). The Raman reporter molecule layer of the secondnanocomposite includes p-aminothiophenol (PATP), and the antibody of thesecond nanocomposite is anti-CD44 antibody. The Raman reporter moleculelayer of the third nanocomposite is p-nitrothiophenol (PNTP), and theantibody of the third nanocomposite is anti-insulin-like growth factor 1receptor antibody (anti-IGF-1). The Raman reporter molecule layer of thefourth nanocomposite comprises 4-(methylsulfanyl)thiophenol (4MSTP), andthe antibody of the fourth nanocomposite is anti-Keratin 18 antibody.

In one embodiment, SERS signal corresponding to each nanocomposite isrepresented by a predetermined color.

In one embodiment, the target of interest includes at least one tumorcell or at least one pathogen.

In one embodiment, the functional molecule is a growth factor thatinduces certain biological functions, including the growth,proliferation of differentiation of cells or organisms.

In one embodiment, the functional molecule is a drug, a virus, a growthfactor, antibiotics, a gene, a plasmid, a vaccine, a plant growth agent,and anti-fungal, a fertilizer, herbicides, or a biological system thatinduces certain biological functions, the death of cells, tissues, ororganisms. The one or more drugs may be anticancer drugs, antibiotics,or antiviral drugs.

In one embodiment, the nanocomposite further includes one or morefluorescent agents. The one or more fluorescent agents can be quantumdots or fluorescent dyes. The one or more fluorescent agents may bemixed with the Raman report molecules and located at the Raman reporterlayer, may be attached to or located at the antibody layer, or may beformed of a separate layer.

In another aspect, the present invention is directed to a system formonitoring conditions of a target of interest. In one embodiment, thesystem includes the nanoagent described above, a surface enhanced Ramanspectrometer configured to provide an incident radiation signal to thetarget of interest and to collect SERS signals generated by the Ramanreporter molecule layer in response to the incident radiation signal,and a processing unit for processing the SERS signals collected by thesurface enhanced Raman spectrometer, so as to monitor the conditions ofthe target of interest.

In a further aspect, the present invention is directed to a method ofmaking at least one nanocomposite for SERS detection, treatment, andmonitoring of a target of interest. In certain embodiments, the methodincludes:

forming at least one gold nanorod;

coating a silver layer on an outer surface of the gold nanorod;

assembling a Raman reporter molecule layer on the coated silver layer,wherein the Raman reporter molecule layer includes Raman reportermolecules detectable by the SERS;

coating a pegylated layer on the assembled Raman reporter moleculelayer; and

conjugating the coated pegylated layer with an active layer.

The active layer includes at least one of a targeting moleculeconfigured to bind to the target of interest and a functional moleculeconfigured to interact with the target of interest.

In one embodiment, the step of forming the at least one gold nanorodincludes:

mixing a first exadecyltrimethylammoniumbromide (CTAB) solution with asilver nitrate solution to form a first mixture;

adding a first HAuCl₄ to the first mixture to form a second mixture;

adding a first ascorbic acid to the second mixture to form a thirdmixture;

adding a seed solution to the third mixture to form a fourth mixture;and

centrifuging the fourth mixture to form a first precipitate, wherein thefirst precipitate comprises the gold nanorod.

In one embodiment, the seed solution is prepared by:

mixing a second CTAB solution with a second HAuCl₄ to form a fifthmixture; and

adding NaBH₄ to the fifth mixture and stirring to form the seedsolution.

In one embodiment, the step of coating the silver layer includes:

dispersing the gold nanorod in a third CTAB solution by sonication toform a sixth mixture;

adding a polyvinylpyrrolidone (PVP) solution and AgNO₃ to the sixthmixture and gently mixing to form a seventh mixture;

adding a second ascorbic acid to the seventh mixture to form an eighthmixture;

adding NaOH solution to the eighth mixture to form a ninth mixture, suchthat the pH of the ninth mixture is elevated to about pH9, and a silverion reduction reaction is initiated; and

centrifuging the ninth mixture to form a second precipitate, wherein thesecond precipitate comprises gold nanorod coated with the silver layer.

In one embodiment, the step of assembling the Raman reporter moleculelayer includes:

dispersing the gold nanorod coated with the silver layer in distilledwater to form a tenth mixture;

dissolving the Raman reporter molecule selected from the groupconsisting of 4-MBA, PATP, PNTP, and 4-MSTP, in ethanol to form areporter solution;

adding the reporter solution to the tenth mixture and stirring for toform an eleventh mixture; and

centrifuging the eleventh mixture to form a third precipitate, whereinthe third precipitate comprises the gold nanorod coated with the silverlayer, and assembled with the Raman reporter molecule layer.

In one embodiment, the step of coating the pegylated layer includes:

dispersing the gold nanorod with the coated silver layer and theassembled Raman report molecule layer in HS-PEG-COOH solution andvigorously stirring to form a twelfth mixture, wherein the HS-PEG-COOHsolution comprises about 2 mg/ml HS-PEG and about 2 mM NaCl;

adding HS-PEG to the twelfth mixture and keep at about 5° C. overnightto form a thirteenth mixture; and

centrifuging the thirteenth mixture to form a fourth precipitate,wherein the fourth precipitate comprises the gold nanorod coated withthe silver layer, assembled with the Raman reporter molecule layer, andcoated with the thiolated PEG layer.

In one embodiment, the step of conjugating the pegylated-Raman-silvercoated gold nanorod with the targeting molecule includes:

suspending the gold nanorod coated with the silver layer, assembled withthe Raman reporter molecule layer, and coated with the pegylated layerin PBS buffer by sonicating to form a suspending mixture;

adding N-hydroxysuccinimide (NHS) and1N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) tothe suspending mixture and stirring to form a fourteenth mixture;

washing the fourteenth mixture by centrifuging twice using PBS buffer toobtain a fourth precipitate;

dispending the fourth precipitate in PBS buffer to form a fifteenmixture;

adding the molecules of the antibody to the fifteenth mixture and mixingthoroughly to form a sixteen mixture, wherein the antibody includesanti-EpCAM, anti-CD44, anti-IGF-1 Receptor β, anti-Keratin 18, and oneor more antibodies specific to the target of interest; and

stirring the sixteenth mixture at room temperature to form thenanocomposite.

In one embodiment, SERS signal corresponding to each nanocomposite ischaracterized with a predetermined color.

In one embodiment, a method of making a nanoagent is provided, and thenanoagent includes one or more nanocomposite produced by the methoddescribed above.

In certain embodiments, the method further includes attaching one ormore molecules of interest to the pegylated layer or the active layer.

In one embodiment, the method further includes attaching one or morefluorescent agents to the nanocomposite. The one or more fluorescentagents may be quantum dots or fluorescent dyes. The one or morefluorescent agents may be mixed with the Raman report molecules beforethe assemble step, so that the assembled Raman reporter molecule layercontains the one or more fluorescent agents. In other embodiments, theone or more fluorescent agents may also be attached to or located at theantibody layer, or may be formed of a separate layer.

In yet another aspect, the present invention is directed to ananocomposite. In one embodiment, the nanocomposite includes ananostructure formed by at least one nanomaterial, and an active layerconjugated to the nanostructure. The active layer has at least one of atargeting molecule configured to bind to a target of interest and afunctional molecule configured to interact with the target of interest.

In one embodiment, the nanostructure has a spherical shape, a tubularshape, a cylindrical shape, or a rod-like triangular shape.

In certain embodiments, the nanomaterial includes silver coated goldnanorods, quantum dots, nanowires, nanotubes, and fullerenes. In oneembodiment, the nanomaterial includes nanomaterials of gold, silver,copper, iron, Fe_(x)O_(y) TiO₂, SiO₂, and carbon.

In certain embodiments, the target of interest is a cancer cell, apathogen, or a plant, and the functional molecule is a virus, a phage, adrug, a growth factor, antibiotics, a gene, a plasmid, a vaccine, aplant growth agent, an anti-fungal, a fertilizer, herbicides, anantibody that specifically binds to S100B, or other biological activemolecules. In one embodiment, the target of interest is the cancer cell,and the functional molecule is the virus that is capable of reproducingin the cancer cell, such that the cancer cell is disrupted or the virusinduces apoptosis of the cancer cell. In one embodiment, the target ofinterest is the plant, and the functional molecule is the plant growthfactor that is capable of promoting growth of the plant. In oneembodiment, the active layer includes a biomarker of TBI or an antibodythat specifically binds to the biomarker. In one embodiment, thebiomarker of TBI includes at least one of S100B, NSE, MBP, caspase-3,interleukins, tau protein, NEFL, NEFH, glial fibrillary acidic protein,APP, and amyloid.

In one embodiment, the functional molecule is further configured tospecifically bind to the target of interest. In this example, thefunctional molecule has the function of both targeting the target ofinterest and treating the target of interest.

In certain embodiments, the nanomaterial includes a core and a shellsurrounding the core. In one embodiment, the core includes at least onegold nanorod, and the shell is a silver layer comprising silvernanoparticles.

In certain embodiments, the gold nanorod has an aspect ratio (AR) in arange of about 1-9, a length in a range of about 10-100 nm, a diameterin a range of about 1-40 nm, and the silver layer has a thickness in arange of about 0.5-5 nm.

In certain embodiments, the nanocomposite further includes a reporterlayer disposed between the nanomaterial and the active layer. Thereporter layer is detectable by at least one of surface enhanced Ramanspectroscopy (SERS), magnetic resonance imaging (MRI), x-rayradiography, computed tomography (CT), positron emissiontomography-computed tomography (PET-CT), and infrared spectroscopy (IR).

In one embodiment, the reporter layer include reporter molecules, andthe reporter molecules include 4MBA, PATP, PNTP, 4MSTP, or othermolecules with unique Raman spectra and intense Raman peak intensities.

In certain embodiments, the nanocomposite further includes a pegylatedlayer disposed between the reporter layer and the active layer, and thepegylated layer has at least one of HS-PEG, HS-PEG-COOH and HS-PEG-NHx.

In certain embodiments, the nanocomposite further includes an activelayer conjugated to the pegylated layer. The active layer includes atleast one of a targeting molecule configured to bind to the target ofinterest and a functional molecule configured to interact with thetarget of interest. In one embodiment, the targeting molecules aremolecules of anti-EpCAM antibody, anti-CD44 antibody, anti-IGF-1antibody, or anti-Keratin 18 antibody, or one or more antibodiesspecific to the target of interest.

In certain embodiments, the functional molecule is conjugated to atleast one of the pegylated layer and the targeting molecule.

In one embodiment, the functional molecule is a growth factor thatinduces certain biological functions, including the growth,proliferation of differentiation of cells or organisms.

In one embodiment, the functional molecule is a virus, a phage, a drug,a growth factor, antibiotics, a gene, a plasmid, a vaccine, a plantgrowth agent, an anti-fungal, a fertilizer, herbicides, an antibody thatspecifically binds to S100 calcium-binding protein B (S100B), or otherbiological active molecules, the death of cells, tissues, or organisms.The one or more drugs may be anticancer drugs, antibiotics, or antiviraldrugs.

In one embodiment, the nanocomposite further includes one or morefluorescent agents. The one or more fluorescent agents can be quantumdots or fluorescent dyes. The one or more fluorescent agents may bemixed with the other report molecules and located at the reporter layer,may be attached to or located at the active layer, or may be formed of aseparate layer.

In one embodiment, the present invention is directed to a nanoagentincluding at least one nanocomposite as described above, for detecting,treating, and monitoring at least one tumor cell or at least onepathogen.

In a further aspect, the present invention is directed to a system fordetecting, treating and monitoring a target of interest. In certainembodiments, the system includes a nanoagent having at least onenanocomposite as described above, a surface enhanced Raman spectrometer,and a processing unit. The surface enhanced Raman spectrometer isconfigured to provide an incident radiation signal to the target ofinterest, and to collect SERS signals generated by the Raman reportermolecule layer in response to the incident radiation signal. Theprocessing unit is configured for processing the SERS signals collectedby the surface enhanced Raman spectrometer, so as to determine whetherthe target of interest has at least one tumor cell or at least onepathogen.

In a further aspect, the present invention is directed to a method ofmaking the nanocomposite as described above. In one embodiment, themethod includes:

providing the nanomaterial;

forming the nanostructure from the nanomaterial; and

conjugating at least one of a targeting molecule and a functionalmolecule to the nanostructure to form the nanocomposite.

Further areas of applicability of the invention will become apparentfrom the detailed description provided hereinafter. It should beunderstood that the detailed description and specific examples areintended for purposes of illustration only and are not intended to limitthe scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the invention. Theinvention may be better understood by reference to one or more of thesefigures in combination with the detailed description of specificembodiments presented herein. The drawings described below are forillustration purposes only. The drawings are not intended to limit thescope of the present teachings in any way.

FIG. 1 schematically shows a nanocomposite of a nanoagent according toone embodiment of the invention.

FIG. 2A shows a flowchart of producing nanocomposites of a nanoagentaccording to one embodiment of the invention.

FIG. 2B schematically shows a process of producing nanocomposites of ananoagent according to one embodiment of the invention.

FIG. 3 shows images and diagrams of gold nanorods, silver coated goldnanorods, and nanocomposites according to certain embodiments of thepresent invention, where (A) shows HRTEM images of gold nanorods andsilver coated gold nanorods according to certain embodiments of thepresent invention; (B) shows SEM and STEM images of a silver coated goldnanorod according to one embodiment of the present invention; (C) showsUV-Visible spectra of gold nanorods and silver coated gold nanorodsaccording to one embodiment of the present invention; and (D) showsRaman signal intensity of gold nanorods, silver coated gold nanorods andnanocomposites having 4MBA according to one embodiment of the presentinvention.

FIG. 4 schematically shows diagrams of preparing and using differenttypes of SERS nanocomposites for cancer cells detection according tocertain embodiments of the invention, where (A) shows a schematicdiagram of preparing four types of SERS nanocomposites and the Ramanspectra (acquisition time 50 seconds) corresponding to each of the fourtypes of SERS nanocomposites according to one embodiment of the presentinvention; (B) shows schematically nanocomposites specifically targetingthe surface of a breast cancer cell to obtain the SERS thermal spectraaccording to one embodiment of the present invention; and (C) showsschematic views that different types of SERS nanocomposites accumulateon the surface of breast cancer cells (MCF-7) and produce multi-colorspectra according to one embodiment of the present invention.

FIG. 5A shows SEM images with EDS elemental analysis of nanocompositeson the MCF-7 cell surface according to one embodiment of the presentinvention.

FIG. 5B shows TEM images of nanocomposites cluster on the surface of anMCF-7 cell according to one embodiment of the present invention.

FIG. 6 shows the immunocytochemistry staining (ICC) of MCF-7 cells inmixed culture with fibroblast cells BJ-1 according to one embodiment ofthe present invention.

FIG. 7A shows Raman mapping images of cells according to certainembodiments of the present invention, where (a1) shows Raman mappingimages for targeting a single MCF-7 cancer cell among fibroblast cellswith four different SERS nanoagents; (a2) shows Raman mapping images ofa cancer cell without using any SERS nanoagents; and (a3) shows Ramanmapping images of a fibroblast cell (normal cell) with four SERSnanoagents.

FIG. 7B shows Raman mapping images of cells according to certainembodiments of the present invention, where (b1) shows Raman mappingimages of a single cancer cell, MCF-7, among millions of white bloodcells, using a nanoagent having four types of nanocomposites; (b2) showsRaman mapping images of a single cancer cell, MCF-7, among millions ofwhole blood cells, using a nanoagent having four types ofnanocomposites; and (b3) shows SERS mapping signal collected from thewhite blood cells only, i.e., without presence of cancer cells, using ananoagent having four types of nanocomposites.

FIG. 7C shows SERS signal collected with different time periods from 1second(s) to 5 s, using a nanoagent having four types of nanocompositesaccording to one embodiment of the present invention.

FIG. 7D schematically shows SERS linear scanning position and SERSsignal of a selected single cancer cell according to one embodiment ofthe present invention, where (d1) shows SERS linear scanning position ofa selected single cancer cell, and (d2) shows SERS signal of a selectedsingle cancer cell scanned linearly four times along the line in (d1),each time with a specific scanning range corresponding to one of thefour types of SERS nanocomposites.

FIG. 8 shows images of SERS signal of a mixture of MCF-7 cell and whiteblood cells, using the negative nanocomposite having CD45, according tocertain embodiments of the present invention.

FIG. 9A shows incorporation of the anticancer drug doxorubicin to ananostructure to form a nanocomposite according to certain embodimentsof the present invention.

FIG. 9B shows UV-Visible spectra of the nanocomposite of FIG. 9A.

FIG. 10A shows incorporation of the herbicedes picloram to ananostructure to form a nanocomposite according to certain embodimentsof the present invention.

FIG. 10B shows UV-Visible spectra of the nanocomposite of FIG. 10A.

FIG. 10C shows incorporation of the herbicedes dicamba to ananostructure to form a nanocomposite according to certain embodimentsof the present invention.

FIG. 10D shows incorporation of the plant hormone 3-indolylacetic acidto a nanostructure to form a nanocomposite according to certainembodiments of the present invention.

FIG. 10E shows incorporation of the plant hormone gibberellic acid to ananostructure to form a nanocomposite according to certain embodimentsof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described more fully hereinafter withreference to the accompanying drawings, in which exemplary embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Likereference numerals refer to like elements throughout.

DEFINITIONS

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. For convenience, certainterms may be highlighted, for example using italics and/or quotationmarks. The use of highlighting and/or capital letters has no influenceon the scope and meaning of a term; the scope and meaning of a term arethe same, in the same context, whether or not it is highlighted and/orin capital letters. It will be appreciated that the same thing can besaid in more than one way.

Consequently, alternative language and synonyms may be used for any oneor more of the terms discussed herein, nor is any special significanceto be placed upon whether or not a term is elaborated or discussedherein. Synonyms for certain terms are provided. A recital of one ormore synonyms does not exclude the use of other synonyms. The use ofexamples anywhere in this specification, including examples of any termsdiscussed herein, is illustrative only and in no way limits the scopeand meaning of the invention or of any exemplified term. Likewise, theinvention is not limited to various embodiments given in thisspecification.

It will be understood that when an element is referred to as being “on”another element, it can be directly on the other element or interveningelements may be present therebetween. In contrast, when an element isreferred to as being “directly on” another element, there are nointervening elements present. As used herein, the term “and/or” includesany and all combinations of one or more of the associated listed items.

It will be understood that, although the terms first, second, third,etc. may be used herein to describe various elements, components,regions, layers and/or sections, these elements, components, regions,layers and/or sections should not be limited by these terms. These termsare only used to distinguish one element, component, region, layer orsection from another element, component, region, layer or section. Thus,a first element, component, region, layer or section discussed below canbe termed a second element, component, region, layer or section withoutdeparting from the teachings of the invention.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising”, or “includes” and/or “including” or “has” and/or“having” when used in this specification specify the presence of statedfeatures, regions, integers, steps, operations, elements, and/orcomponents, but do not preclude the presence or addition of one or moreother features, regions, integers, steps, operations, elements,components, and/or groups thereof.

Furthermore, relative terms, such as “lower” or “bottom” and “upper” or“top”, may be used herein to describe one element's relationship toanother element as illustrated in the figures. It will be understoodthat relative terms are intended to encompass different orientations ofthe device in addition to the orientation shown in the figures. Forexample, if the device in one of the figures is turned over, elementsdescribed as being on the “lower” side of other elements would then beoriented on the “upper” sides of the other elements. The exemplary term“lower” can, therefore, encompass both an orientation of lower andupper, depending on the particular orientation of the figure. Similarly,if the device in one of the figures is turned over, elements describedas “below” or “beneath” other elements would then be oriented “above”the other elements. The exemplary terms “below” or “beneath” can,therefore, encompass both an orientation of above and below.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art and theinvention, and will not be interpreted in an idealized or overly formalsense unless expressly so defined herein.

It will be understood that when an element is referred to as being “on”,“attached” to, “connected” to, “coupled” with, “contacting”, etc.,another element, it can be directly on, attached to, connected to,coupled with or contacting the other element or intervening elements mayalso be present. In contrast, when an element is referred to as being,for example, “directly on”, “directly attached” to, “directly connected”to, “directly coupled” with or “directly contacting” another element,there are no intervening elements present. It will also be appreciatedby those of skill in the art that references to a structure or featurethat is disposed “adjacent” to another feature may have portions thatoverlap or underlie the adjacent feature.

As used herein, “around”, “about”, “substantially” or “approximately”shall generally mean within 20 percent, preferably within 10 percent,and more preferably within 5 percent of a given value or range.Numerical quantities given herein are approximate, meaning that theterms “around”, “about”, “substantially” or “approximately” can beinferred if not expressly stated.

As used herein, the terms “comprise” or “comprising”, “include” or“including”, “carry” or “carrying”, “has/have” or “having”, “contain” or“containing”, “involve” or “involving” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to.

As used herein, the term “MCF-7” refers to a breast cancer cell lineisolated in 1970 from a 69-year-old Caucasian woman. MCF-7 is theacronym of Michigan Cancer Foundation-7, referring to the institute inDetroit where the cell line was established in 1973 by Herbert Soule andco-workers. The Michigan Cancer Foundation is now known as the BarbaraAnn Karmanos Cancer Institute. Prior to MCF-7, it was not possible forcancer researchers to obtain a mammary cell line that was capable ofliving longer than a few months. The patient, whose name, FrancesMallon, is unknown to the vast majority of cancer researchers, died in1970. Her cells were the source of much of current knowledge aboutbreast cancer. At the time of sampling, she was a nun in the convent ofImmaculate Heart of Mary in Monroe, Mich. under the name of SisterCatherine Frances. MCF-7 and two other breast cancer cell lines, namedT-47D and MDA-MB-231, account for more than two-thirds of all abstractsreporting studies on mentioned breast cancer cell lines, as concludedfrom a Medline-based survey.

As used herein, the term “BJ-1 cell line” refers to a normal skinfibroblast cell line, which is available from American Type CultureCollection (ATCC) with ATCC number CRL-2522.

As used herein, the term “circulating tumor cells” or “CTCs” refers tocells that have shed into the vasculature from a primary tumor andcirculate in the bloodstream. CTCs thus constitute seeds for subsequentgrowth of additional tumors (metastasis) in vital distant organs,triggering a mechanism that is responsible for the vast majority ofcancer-related deaths.

As used herein, the term “DMEM” refers to Dulbecco's Modified EagleMedium, and EMEM is the abbreviation of Eagle's Minimum EssentialMedium.

As used herein, the term “ICC” refers to the abbreviation ofimmunocytochemistry staining.

As used herein, the term “4MBA” refers to 4-mercaptobaezoic acid, PNTPis the abbreviation of p-nitrobenzoic acid, PATP is the abbreviation ofp-aminobenzoic acid, 4MSTP is the abbreviation of 4-methylsulfanylthiophenol, and 4APDS is the abbreviation of 4-aminophenyldisulfide.

As used herein, the term “HS-PEG-COOH and HS-PEG” refer to thiolatedpolyethylene glycol with or without acid terminal, respectively.

As used herein, the term “phosphate buffered saline” or “PBS” refers toa buffer solution commonly used in biological research. It is awater-based salt solution containing sodium phosphate, sodium chlorideand, in some formulations, potassium chloride and potassium phosphate.The osmolarity and ion concentrations of the solutions match those ofthe human body (isotonic).

As used herein, the term “bovine serum albumin” or “BSA” or “Fraction V”refers to a serum albumin protein derived from cows. It is often used asa protein concentration standard in lab experiments.

As used herein, the term “fetal bovine serum” or “FBS” or “fetal calfserum” refers to the blood fraction remaining after the naturalcoagulation of blood, followed by centrifugation to remove any remainingred blood cells. Fetal bovine serum comes from the blood drawn from abovine fetus via a closed system of collection at the slaughterhouse.Fetal bovine serum is the most widely used serum-supplement for the invitro cell culture of eukaryotic cells. This is due to it having a verylow level of antibodies and containing more growth factors, allowing forversatility in many different cell culture applications.

OVERVIEW OF THE INVENTION

The conjugation of the nanomaterials with targeting molecules such asantibodies, folates, aptamer or immune protein could provide specificdelivery of the nanomaterials to various cancer cell lines, withinminutes [3-8]. Recently, quantum dot nanomaterials and iron oxidenanoparticles have been used widely as imaging and diagnostic nanoagentsfor cancer cells [16-18]. Among these new and enhanced imaging anddiagnostic assays, surface-enhanced Raman spectroscopy (SERS) has beenstudied and proposed for early imaging and detection [19-22] of tumorcells.

In one aspect, the present invention is directed to a biocompatiblenanoagent for detecting a target of interest by SERS, and optionallytreating the target of interest using functional molecules attached tothe nanoagent, and monitoring the conditions of the target of interestby SERS. In certain embodiments, the target of interest may include atleast one tumor cell, at least one pathogen, or a plant. The tumor cellcan be a benign tumor cell or a malignant tumor cell. The malignanttumor cell, or a cancer cell, can be located locally or a circulatingtumor cell (CTC). The pathogen can be a virus, bacterium, prion, fungusor protozoan that causes disease in its host. In certain embodiments,the nanoagent includes at least one nanocomposite, such as fourdifferent types of nanocomposites.

In one embodiment, the nanocomposite includes at least one gold nanorod,a silver layer coated on an outer surface of the gold nanorod and havingsilver nanoparticles, a Raman reporter molecule layer coated on thesilver layer and having Raman reporter molecules, a pegylated layercoated on the Raman reporter molecule layer and having at least one ofthiolated polyethylene glycol (HS-PEG), thiolated polyethylene glycolacid (HS-PEG-COOH) and HS-PEG-NHx, and an active layer conjugated to thepegylated layer. The active layer includes at least one of a targetingmolecule configured to bind to a target of interest and a functionalmolecule configured to interact with the target of interest. Thefunctional molecule may include virus, drugs, growth factors,antibiotics, genes, plasmids, vaccines, plant growth agents,anti-fungal, fertilizer, herbicides, an antibody that specifically bindsto S100 calcium-binding protein B (S100B), or other biological activemolecules, that are capable of interacting with the target of interest.In one embodiment, the AR of the gold nanorod is in a range of 2-5. Inone embodiment, the AR of the gold nanorod is in a range of 2.77-3.23 oris about 3±0.23, and the length and the diameter of the gold nanorod isin a range of about 35.20-36.80 and about 11.59-12.41 respectively, orabout 36±0.80 nm and about 12±0.41 nm, respectively, and the thicknessof the silver layer is about 1-2 nm, or about 1.7 nm.

In one embodiment, the at least one nanocomposite includes a firstnanocomposite, a second nanocomposite, a third nanocomposite, and afourth nanocomposite. The Raman reporter molecule layer of the firstnanocomposite includes 4-mercaptobenzoic acid (4MBA), and the antibodyof the first nanocomposite is anti-epithelial cell adhesion moleculeantibody (anti-EpCAM). The Raman reporter molecule layer of the secondnanocomposite includes p-aminothiophenol (PATP), and the antibody of thesecond nanocomposite is anti-CD44 antibody. The Raman reporter moleculelayer of the third nanocomposite includes p-nitrothiophenol (PNTP), andthe antibody of the third nanocomposite is anti-insulin-like growthfactor 1 receptor antibody (anti-IGF-1). The Raman reporter moleculelayer of the fourth nanocomposite includes 4-(methylsulfanyl)thiophenol(4MSTP), and the antibody of the fourth nanocomposite is anti-Keratin 18antibody.

In one embodiment, SERS signal corresponding to each nanocomposite isrepresented by a predetermined color, such that SERS signals of thenanoagent having multiple nanocomposites are represented by multiplecolors.

In another aspect, the present invention is directed to a system fordetecting, treating, and monitoring a target of interest. In certainembodiments, the target of interest includes at least one tumor cell orat least one pathogen. In certain embodiments, the system includes ananoagent as described above that has multiple nanocomposites, a surfaceenhanced Raman spectrometer configured to provide an incident radiationsignal to the target of interest, and to collect surface enhanced Ramanspectroscopy (SERS) signals generated by the Raman reporter moleculelayer in response to the incident radiation signal, and a processingunit for processing the SERS signals collected by the surface enhancedRaman spectrometer to determine whether the target of interest have atleast one tumor cell or at least one pathogen, and to monitor the changeof the target of interest after treatment of the target of interest bythe functional molecule.

In a further aspect, the present invention is directed to a method ofmaking a nanocomposite or a nanoagent having one or more nanocomposites,where the nanocomposite/nanocomposites have the structures as describedabove, and the nanocomposite/nanoagent can be used for surface enhancedRaman spectroscopy (SERS) detection and monitoring of a target ofinterest, such as at least one tumor cell or at least one pathogen. Inone embodiment, the nanoagent includes functional molecules that can beused to treat the target of interest. In one embodiment, the nanoagentcan be used for monitoring the change of the target of interest by SERSduring or after treatment of the target of interest using the functionalmolecules.

In one embodiment, the method includes: forming a gold nanorod, coatinga silver layer on an outer surface of the gold nanorod; assembling aRaman reporter molecule layer on the coated silver layer, wherein theRaman reporter molecule layer includes Raman reporter molecules that aredetectable by the SERS; coating a pegylated layer on the assembled Ramanreporter molecule layer; and conjugating the coated pegylated layer withtargeting molecules and/or functional molecules to form the active layerto make the nanocomposite.

Anti-EpCam antibody, anti-CD44 antibody, anti-keratin 18 antibody,anti-IGF-I antibody, or one or more antibodies specific to the target ofinterest are used in the present invention as the targeting molecules.

Since the epithelial cell adhesion molecule (EpCam) antigen is highlyexpressed in normal epithelial cells and the MCF-7 cells originatingfrom these cells, the cells express a considerable amount of EpCamantigen on their surface. Because the EpCam antigen is greatlyover-expressed in many types of cancers, including colon, hepatic,pancreatic, prostate, and breast cancer [32], Anti-EpCam has been usedextensively in breast cancer detection.

CD44 is a cell-cell and cell-matrix adhesion molecule known to be highlyexpressed in many types of cancers and widely used in the diagnosis andprognosis of breast cancer [33, 34]. CD44 is important in tumordevelopment and progression, and anti-CD44 provides multiple prospectsfor advanced cancer treatments by targeting therapeutics to the CD44receptor of the metastasizing tumors, interfering with the CD44signaling pathway.

Keratin 18 is known to be highly expressed in normal mammary epithelialcells, and MCF-7 cells are adenocarcinoma derived from simple breastepithelium. In one embodiment, anti-keratin 18 antibodies have been usedin diagnostic histopathology of breast cancer. In addition, it has beenshown that the down-regulation of membrane keratin 18 plays a key rolein the prognosis of the breast cancer patient [35].

Insulin-like growth factor 1 (IGF-I) is expressed in 90% of breastcancer specimens. Therefore, the anti-IGF-I antibody is used as anmolecule to target breast cancer cells [36, 37].

These and other aspects of the invention are more specifically describedbelow.

IMPLEMENTATIONS AND EXAMPLES OF THE INVENTION

Without intend to limit the scope of the invention, further exemplaryprocedures and preliminary experimental results of the same according tothe embodiments of the invention are given below.

In one aspect, the present invention is directed to a biocompatiblenanoagent for detecting, and monitoring a target of interest, such as atleast one cancer cell or at least one pathogen by SERS, and treating thetarget of interest by the functional molecule attached to the nanoagent.In certain embodiments, the biocompatible nanoagent includes one or morenanocomposites.

FIG. 1 schematically shows a nanocomposite of a nanoagent according toone embodiment of the invention. Referring to FIG. 1, each of thenanocomposite 100 includes a core 102, a shell 106 wrapped around thecore 102, a reporter layer 110 assembled on the shell 106, a bindinglayer 114 coated on the reporter layer 110, and an active layer 118conjugated to the binding layer 114.

In certain embodiments, the core 102 is a gold nanorod (AuNR). Theaspect ratio (AR) is defined as the ratio of the length of the AuNR tothe diameter of the AuNR. In one embodiment, the AR of the AuNR 102 maybe in the range of about 0.3-30, and the length and diameter of the AuNR102 may be in the range of about 3.6-360 nanometer (nm) and about1.2-120 nm, respectively. In one embodiment, the AR of the AuNR 102 isin the range of about 1-9. In one embodiment, the precise AR of the AuNR102 is in the range of about 2-5. In one embodiment, the precise AR ofthe AuNR 102 is in the range of about 2.77-3.23, or about 3±0.23. In oneembodiment, the length and diameter of the AuNR 102 may be in the rangeof about 10-100 nm and about 1-40 nm, respectively. In one embodiment,the particle length and diameter of the AuNR 102 may be approximately36±0.80 nm and 12±0.41 nm, respectively. In one embodiment, these twodimensions are adequate to form two kinds of surface plasmon modes: aweak one around 520 nm transvers mode, and a very strong longitudinalplasmon around 766 nm [26]. The longitudinal surface plasmon is crucial,and the maximum excitation of this strong surface plasmon mode can beachieved when excited by a Raman excitation laser at about 784 nm. Thisensures ultimate sensitivity and very low detection limits when usesSERS for cancer cell detection.

In one embodiment, the shell 106 is a silver layer. The silver layer 106is coated on the AuNR 102 to form a silver coated gold nanorod(AuNR/Ag). In one embodiment, the AuNR 102 and the silver layer 106 haverough surfaces.

In one embodiment, the thickness of the silver layer 106 may be in therange of about 0.2-20 nm. In one embodiment, the thickness of the silverlayer 106 is in the range of about 0.5-5 nm. In one embodiment, thethickness of the silver layer 106 is about 1-2 nm. In one embodiment,the thickness of the silver layer 106 is about 1.7 nm. The thin silverlayer 106 helps maintain the longitudinal surface plasmon wavelength asclose as possible to the excitation laser source (784 nm), in order toachieve the maximum SERS signal. Any thick silver coating will changethe surface plasmon significantly [30].

In one embodiment, the reporter layer 110 is a Raman reporter moleculelayer having Raman reporter molecules. In one embodiment, the Ramanreporter molecules are thiolated organic molecules absorbed on thesurface of the AuNR/Ag. In one embodiment, the Raman reporter moleculemay be at least one of 4-mercaptobenzoic acid (4MBA), p-aminothiophenol(PATP), p-nitrothiophenol (PNTP), 4-(methylsulfanyl)thiophenol (4MSTP),and other molecules with unique Raman spectra and intense Raman peakintensities. In other words, the one or more nanocomposites 100 of thenanoagent may include at least one of the following four types ofnanocomposites: a nanocomposite having a 4MBA reporter layer, ananocomposite having a PATP reporter layer, a nanocomposite having aPNTP reporter layer, and a nanocomposite having a 4MSTP reporter layer.In certain embodiments, the nanoagent may include all of these fourtypes of nanocomposites 100. All the SER Raman spectra are obtainedthrough the detection of those Raman reporter molecules.

In the above embodiment, the reporter molecule is a Raman reportermolecule. In certain embodiments, the reporter layer 110 may includeother type of reporter molecules such that the produced nanoagent may beused together with detecting methods other than SERS, such as MRI, x-rayradiography, CT or IR. In certain embodiments, the reporter molecule isdetectable by different methods. In certain embodiments, the reportmolecules may include one or more fluorescent agents. The one or morefluorescent agents can be quantum dots or fluorescent dyes.

In the above embodiment, the nanoagent includes at least one of the fourtypes of nanocomposites corresponding to four types of reportermolecules. In certain embodiments, the nanoagent may include all fourtypes of nanocomposites. In certain embodiments, the nanoagent mayinclude one, two, three, or more than four types of nanocomposites, andeach type of nanocomposite has a special type of reporter molecule. Inother embodiments, one type of nanocomposite may include two or moredifferent types of reporter molecules. In certain embodiments, one typeof nanocomposite may also include two, three, four or more types ofreporter molecules.

In certain embodiments, the nanocomposite may not include the reporterlayer 110. For example, if detection and monitoring of the target ofinterest have been done, or the detection and monitoring of the targetof interest are not necessary, the nanocomposite does not have toinclude the reporter layer 110. In one embodiment, the binding layer 114is directly applied to the shell layer 106.

In one embodiment, the binding layer 114 is applied to the surface ofthe SERS reporter molecule coated AuNR/Ag. In one embodiment, thebinding layer 114 is a pegylated layer. In one embodiment, the pegylatedlayer may include thiolated PEG polymers, for example, at least one ofHS-PEG, HS-PEG-COOH and HS-PEG-NHx, which are suitable for being used asSERS tags and are non-toxic. Additionally, the thiolated PEG polymers donot displace Raman reporter molecules, which attach to the surface ofgold nanoparticles [40]. In certain embodiments, the x in the HS-PEG-NHxis a positive integer. In one embodiment, x is 1 or 2.

In one embodiment, the pegylated layer 114 includes a mixture of HS-PEGand HS-PEG-COOH, which serves as protective, bio-dispersive and linkerto the later conjugated antibodies. In one embodiment, the averagemolecular weight of the HS-PEG is about 5 kD, and the average molecularweight of the HS-PEG-COOH is about 3 kD. In one embodiment, each nanorod(SERS reporter molecule coated AuNR/Ag) requires about 4,200 moleculesto assure complete surface coverage, i.e. each HS-PEG molecule required0.35 nm² footprint [39]. The pegylated layer 114 may achieve at leasttwo purposes. First, the pegylated layer 114 protects the nanorodssurface and makes the nanocomposite more hydrophilic, and easilydisperses the nanocomposite in aqueous medium, for example, biologicalfluids. Second, the pegylated layer 114 provides a carboxylic terminalon the surface of the SERS reporter molecule coated AuNR/Ag, which isthe linker between the SERS reporter molecule coated AuNR/Ag surface andthe antibodies that will attached thereon for targeting the target, suchas cancer cells.

In certain embodiments, as described above, the pegylated layer 114 maybe coated on the shell 106 directly, and the reporter layer 110 is notnecessary. In one embodiment, the nanocomposite 100 does not include thepegylated layer 114 and the reporter layer 110, and the active layer 118is directly attached to the shell 106.

In certain embodiments, the active layer 118 includes at least one of atargeting molecule and a functional molecule. The targeting molecule isconfigured to specifically guide the nanoagent to the target of interestand specifically binds to the target of interest. The functionalmolecule is able to interact with the target of interest. In certainembodiments, the targeting molecule includes antibodies. The antibody ofthe active layer 118 includes molecules of a type of antibody whichspecifically targeting certain cancer cell surface antigen. In oneembodiment, the antibody is attached covalently to HS-PEG-COOH (—COOHterminal) and plays a role in the specific SERS nanocomposite deliveryto the cancer cells.

In one embodiment, the antibody of the active layer 118 may includemolecules of at least one of an anti-EpCAM antibody, an anti-CD44antibody, an anti-IGF-1 Receptor β antibody, an anti-Keratin 18antibody, and one or more antibodies specific to the target of interest.In other words, the one or more nanocomposites 100 of the nanoagent mayinclude at least one of the following four types of nanocomposites: thenanocomposite having an anti-EpCAM antibody layer, the nanocompositehaving an anti-CD44 antibody layer, the nanocomposite having ananti-IGF-1 Receptor β antibody layer, and the nanocomposite having ananti-keratin 18 antibody layer. In one embodiment, the biocompatiblenanoagent having at least one of the four types of nanocomposites may beused for detecting and imaging breast cancer cells, for example, MCF-7,and allow for the capability to distinguish one single cancer cellsamong normal cells. In one embodiment, the biocompatible nanoagentincludes all four types of nanocomposites.

In the above embodiment, the targeting molecule includes antibodies. Incertain embodiments, the targeting molecule may include other type oftargeting molecules to specifically binding an object, for example, aligand that can bind a receptor, or a lectin that can bind acarbohydrate.

In certain embodiments, the nanoagent may not include the targetingmolecule in the active layer 118. The nanoagent may circulate in apatient's body or a plant, and thus is able to in contact with thetarget of interest.

In certain embodiments, at least one of the reporter layer 110 and thebinding layer 114 is not present in the nanocomposite 100. The activelayer 118 thus is attached to the shell 106 or the reporter layer 110.

In certain embodiments, the active layer 118 includes functionalmolecules, and the functional molecules are attached to at least one ofthe binding layer 114 and the targeting molecules in the active layer118. For example, the targeting molecules in the active layer 118 may beattached to certain amount of surfaces of the binding layer 114, whilethe functional molecules in the active layer 118 occupy certain amountof surfaces of the binding layer 114. The targeting molecules and thefunctional molecules altogether may occupy the complete surface of thebinding layer 114 to have efficient binding and treating effects. In oneembodiment, the targeting molecules and the functional moleculesaltogether may only occupy parts of the outer surface of the bindinglayer, as long as the nanoagent provides efficient binding and treatmentto the target of interest. In certain embodiments, the targetingmolecules and the functional molecules may be attached to each other bychemical bond, hydrophobic force, van der Waals force, or any otherinteraction forces, and at least one of the targeting molecule and thefunctional molecule is attached to the binding layer 114. The attachmentof the targeting molecules and the functional molecules may be in anysequence. In one embodiment, the targeting molecules and the functionalmolecules may be mixed and attached to the binding layer 114 at the sametime. In one embodiment, the targeting molecules are attached to thebinding layer 114 to form a first layer, and the functional moleculesare attached to the first layer of the targeting molecules to form asecond layer of functional molecules. Alternatively, the functionalmolecules are attached to the binding layer 114 to form a first layer,and the targeting molecules are attached to the first layer of thefunctional molecules to form a second layer of the targeting molecules.In other words, the active layer 118 may be implemented by a singlelayer or multiple layers. Further, at least a portion of the targetingmolecules and a portion of the functional molecules need to be exposedin the surface of the nanocomposite to efficiently fulfill theirfunction. In other embodiments, at least a portion of the targetingmolecules are exposed to ensure delivering of the nanocomposite to thetarget of interest, and during the delivering and after the delivering,the functional molecules may be exposed or released to fulfill theirfunction.

In certain embodiments, the functional molecules are growth factor thatinduces certain biological functions, including the growth,proliferation of differentiation of cells or organisms. In oneembodiment, the functional molecules are proteins, drug molecules,virus, or a biological system that induces certain biological functions,the death of cells, tissues, or organisms. The one or more drugs may beanticancer drugs, antibiotics, or antiviral drugs. In one embodiment,the functional molecule is a type of virus that specifically binds anddisrupts the target of interest.

In certain embodiments, the functional molecules may function as thetargeting molecules as well, such that the functional molecules not onlyspecifically binds to the target of interest, but also interact with thetarget of interest to accomplish certain function. For example, when thetarget of interest is cancer cells, the functional molecules may bevirus that specifically binds to the cancer cells, disrupts the cancercells, or/and induce the cancer cells to an apoptosis process.

In the above embodiment, the nanoagent may include at least one of thefour types of nanocomposites corresponding to four types of reportermolecules. In certain embodiments, the nanoagent may include all fourtypes of nanocomposites. In certain embodiments, the nanoagent mayinclude one, two, three, or more than four types of nanocomposites, andeach type of nanocomposite has a special type of reporter molecule. Inother embodiments, one type of nanocomposite may include two or moredifferent types of reporter molecules.

In the above embodiment, the nanoagent includes at least one of the fourtypes of nanocomposites corresponding to four types of antibodies. Incertain embodiments, the nanoagent may include all four types ofnanocomposites. In certain embodiments, the nanoagent may include one,two, three, or more than four types of nanocomposites, and each type ofnanocomposite has a specific type of antibody. In other embodiments, onetype of nanocomposite may include two or more different types ofantibodies.

In one embodiment, the nanoagent as described above can be used todetect at least one tumor cell or at least one pathogen by SERS,treating the at least one tumor cell or the at least one pathogen by thefunctional molecules, and monitoring the conditions of the at least onetumor cell or the at least one pathogen by SERS. In one embodiment, thetumor cell is a circulating tumor cell.

In certain embodiments, the functional molecule is a growth factor thatinduces certain biological functions, including the growth,proliferation of differentiation of cells or organisms. In oneembodiment, the molecule of interest is a protein, a drug, or abiological system that induces certain biological functions, the deathof cells, tissues, or organisms. The one or more drugs may be anticancerdrugs, antibiotics, or antiviral drugs. In one embodiment, thefunctional molecule is a virus. The virus may only infect the target ofinterest, such as a cancer cell, without affecting normal cells. In oneembodiment, the virus only grows in the cancer cell. In one embodiment,the virus may hijack the cancer cell's protein making mechanism or thecancer cell's gene copying capabilities, and reproduce the virus in vastnumbers. Once the cancer cell dies, the vast number of virus will bereleased to affect other cancer cells. In one embodiment, when the virusinfects a cancer cell, the virus may reprogram the cancer cell toself-destruct, i.e., induce apoptosis of the cancer cell.

In certain embodiments, the functional molecules of virus also haveseveral surprising mechanisms that go beyond the infected tumor cell.Some strains of oncogenic viruses also infect the tumor blood vessels.This infection attracts immune cells that destroy the vessels and chokeoff the blood supply for the tumor. The body's own immune system isessential for the virus to work. In certain embodiments, as the tumorgrows, the immune system cannot or does not recognize the danger andfails to act. However, killing of cancer cells by the virus producescellular debris that induces the production of small immune stimulatingmolecules call cytokines and also activate the body's immune systemdendritic cells which in turn alert the immune systems T cells to mounta response to the cancer. In one embodiment, the functional molecules ofvirus can be made to selectively replicate in cancer cells whilevirtually ignoring normal cells. Additionally, the viral genome can berevised to give the virus additional cancer fighting traits such as theability to enhance the body's immune system to fight the cancer. Incertain embodiments, once the functional molecules of virus aredelivered to a cancer cell, the virus turns the cancer cell into afactory that reproduces more and more virus till all the host cells(cancer) are gone.

Most cancer cells are really the offspring of one ancestral aberrantcell but now possess a different genetic make-up with enhancedcomplexity. Most cancer chemotherapy meds work through one mechanism.Unfortunately, cancer cells frequently adapt and render the chemouseless. The treatment by the functional molecules of virus is much likecombination chemo, which attacks at multiple sites with less likelihoodof the cancer cell becoming resistant.

The virus may be designed to specifically affect the cancer cells.Cancer cells have unique cellular surface receptors. In certainembodiments, one approach is to engineer viruses to hone in on thesereceptors and not normal cells. Another approach would be to carry thevirus to the receptor with a specific carrier directed to the cancer.For example, the targeting molecules of the nanoagent guide thenanoagent to be delivered to the cancer cell. Accordingly, the virusattached to the binding layer or the targeting molecules is contactableto the cancer cell.

Because cancer cells grow quickly, they are in greater need for the“building blocks” of DNA. Knowing this proclivity, viruses may beengineered such that the viruses are drawn to the excessive amount ofbuilding material found in cancer cell.

As cells undergo malignant transformation, they lose their ability toproduce an antiviral molecule called interferon. This renders them moresusceptible to infection with little ability to remove the virus.

It would appear that the activation of the host immune system is allimportant. Unfortunately, many tumors have immune suppressing cells. Amajor challenge will be to devise a way to shut down these cells toallow the immune system to work more effectively. In certain embodiment,a monoclonal antibody (such as PD-1) may be used as the targetingmolecule so as to deliver the virus to the cancer cell. The virus thenshuts down these cancer cells to allow the immune system to work moreeffectively.

In certain embodiments, the nanoagent may be applied to the target ofinterest many times. For example, once a patient is detected withcancer, a nanoagent having the functional molecules for treating thatcancer is applied to the patient. The dosage and frequency of thenanoagent may be determined according to the conditions of the patient.In one embodiment, the treatment may include applying the nanoagent tothe patient at day 1, day 7, and day 14.

After treatment by the functional molecules of the nanoagent, the targetof interest may be reduced by number or disappear completely. In certainembodiments, the nanoagent of the present invention provides aconvenient and accurate monitoring of the conditions after treatment ofthe target of interest. In one embodiment, for a day 1, day 7 and day 14application of the nanoagent, the conditions of the patient may bechecked before each application of the nanoagent, or monitored atpredetermined intervals, or continuously for a short time whennecessary.

In other embodiments, the nanoagent is configured to detect a target ofinterest other than at least one tumor cell, or configured to be usedwith methods other than SERS. In one embodiment, the nanoagent isconfigured to detect a specific type of cell, for example a cancer cell,a blood and immune system cell, a hormone secreting cell, or any othercells that express a specific antigen or certain cell surface molecules.In one embodiment, the nanoagent is configured to detect a pathogen, forexample a bacteria, a fungus or a virus. In one embodiment, thenanoagent is configured to detect an exogenous chemical or device thatis applied to a patient. In one embodiment, the detection using thenanoagent can be performed in vivo or in vitro.

In certain embodiments, when the target of interest is pathogenicbacteria, the nanocomposite may include phages or bacteriophages in itsactive layer to treat the pathogenic bacteria. In other words, thephages are attached to the outer surface of the nanocomposite and usedas functional molecules. When the nanoagent is delivered to be incontact with the pathogenic bacteria, the phages are capable ofinfecting and replicating in the pathogenic bacteria, and thus killingthe pathogentic bacteria. Those phages are much more specific thanantibiotics. The phages may be chosen to specifically treat thepathogenic bacteria, with no harm to the host organism and otherbeneficial bacteria in the host.

In certain embodiments, the nanoagent may be used to detect traumaticbrain injury (TBI). Evaluate TBI risks, and monitor the conditions of aTBI patient after treatment. TBI is a brain dysfunction caused by anexternal force, usually a violent blow to the head, which often occursin military personnel and athlete. Early detection of TBI or TBI risks,and monitoring the recovery of patients from TBI, may be performed byevaluating the amount of certain biomarkers in body fluid of thepatients, such as the amount of biomarkers in the cerebrospinal fluid orblood. Those biomarkers includes S100 calcium-binding protein B (S100B),neuron-specific enolase (NSE), myelin basic protein (MBP), caspase-3,interleukins, tau protein, neurofilament light polypeptide (NEFL),neurofilament heavy polypeptide (NEFH), glial fibrillary acidic protein,amyloid precursor protein (APP), and amyloid, etc.

In certain embodiments, the nanoagent used for detecting TBI or TBIrisks or monitoring status of the TBI patient may include an antibodyagainst at least one of the above identified TBI biomarkers. Theantibody may be attached to the outer surface of the nanocomposite ofthe nanoagent. In one embodiment, the antibody may be used as thetargeting molecule to deliver certain medicine to the location where thecorresponding biomarker exists in an amount greater than normal. In oneembodiment, the antibody may be used as the functional molecules toneutralize the biomarkers. The nanoagent having the antibody may be usedto measuring concentration of the biomarkers or locating thosebiomarkers in the patient. In one embodiment, the S100B antibody is usedas the targeting molecules or/and functional molecules for detecting thepresence or concentration of S100B, or neutralize the biomarkers, wherethe biomarker may be S100B.

In certain embodiments, the nanoagent may be attached with at least oneof the biomarkers of TBI. Those biomarkers may be used to detectcounterpart components in the TBI patients, so as to detect TBI of thepatient, evaluate TBI risks of the patient, or monitoring status of theTBI patients after treatment. In one embodiment, the biomarker attachedto the nanoagnet is S100B.

In one embodiment, the nanoagent is configured to detect, treat ormonitor a plant. In one embodiment, the functional molecules of thenanoagent include plant growth factors for improving growth of theplant. In certain embodiments, for cost effective treatment of theplant, the nanoagent may only include a nanostructure and a functionalmolecule attached to the nanostructure. The nanostructure may be formedby a nanomaterial of quantum dots, nanowires, nanorods, nanofibers,fullerene, and silver coated gold nanorod, etc. The functional moleculemay be a plant growth factor.

In one embodiment, the nanoagent includes Raman reporter molecules thatare detectable by SERS. In certain embodiment, the nanoagent may includereporter molecules detectable by methods other than SERS, such as MRI,x-ray radiography, CT, or IR. The nanoagent is therefore configured tobe applied with methods other than SERS, for specific targeting,detection, and treatment of cancer cells or other targeted cells,tissues or objects. In certain embodiments, the reporter molecules aredetectable by two, three, four or more different methods describedabove. In certain embodiments, the report molecules may include one ormore fluorescent agents. The one or more fluorescent agents can bequantum dots or fluorescent dyes.

In another aspect, the present invention is directed to a system fordetecting a target of interest and optionally monitoring the conditionsof the target of interest. In one embodiment, the system includes thenanoagent described above, a surface enhanced Raman spectrometer, and aprocessing unit. The surface enhanced Raman spectrometer is configuredto provide an incident radiation signal to the target of interest, andto collect SERS signals generated by the Raman reporter molecule layerin response to the incident radiation signal. The processing unit isconfigure to process the SERS signals collected by the surface enhancedRaman spectrometer, so as to detect the target of interest andoptionally monitor conditions of the target of interest.

In a further aspect, the present invention is directed to a process formaking a biocompatible nanoagent 200. In certain embodiments, thebiocompatible nanoagent 200 may have the structure as shown in FIG. 1.

FIG. 2A shows a flowchart of producing nanocomposites of a nanoagentaccording to one embodiment of the invention. FIG. 2B schematicallyshows a process of producing nanocomposites of a nanoagent according toone embodiment of the invention. Referring to FIGS. 2A and 2B, theprocess of producing nanocomposites includes a plurality of operations.At operation 252, a core (e.g., the AuNR 202) is formed. At operation256, a shell layer (e.g., the silver layer 206) is wrapped around thecore (e.g., the AuNR 202) to form the AuNR/Ag. At operation 260, areporter layer 210 (e.g., M1, M2, M3 or M4) is assembled or coated onthe surface of the shell layer (e.g., the silver layer 206). Atoperation 264, a binding layer 214 (e.g., the pegylated layer) is coatedon the reporter layer 210. At operation 268, an active layer 218 isattached on the binding layer 214.

As discussed above, the core 202 being prepared in operation 252 may bethe AuNR. In one embodiment, the AuNR 202 with tuned size is preparedaccording to the seed mediated method by Nikoobakht [26]. Specifically,5 ml of 0.2 M hexadecyltrimethylammoniumbromide (CTAB) solution is mixedwith 5 ml of 0.0005 M HAuCl₄, and then 600 μl of NaBH₄ is added to themixture with stirring for about two minutes, to form a seed solution. Tosynthesize AuNRs with an aspect ratio around 3, 5 ml of 0.2 M CTAB ismixed with 150 μl of 0.004 M silver nitrate solution to form a firstmixture. Then, 5 ml of 0.001 M HAuCl4 is added to and mixed with thefirst mixture to form a second mixture. After that, 70 μl of 0.0788 Mascorbic acid is mixed with the second mixture to form a third mixture.Finally, 12 μl of the prepared seed solution is added to the thirdmixture to form a fourth mixture. The fourth mixture is kept at 30° C.for about 40 minutes without any further stirring to form the AuNRs.

FIG. 3 (A) shows HRTEM images of gold nanorods and silver coated goldnanorods according to certain embodiments of the present invention, andFIG. 3 (C) shows UV-Visible spectra of gold nanorods and silver coatedgold nanorods according to one embodiment of the present invention. Asshown by HRTEM images of (a1) and (a2) of FIG. 3 (A), the particlelength and diameter of the AuNRs are approximately 36±0.80 nm and12±0.41 nm, respectively. As shown in FIG. 3 (C), these two dimensionsare adequate to form two kinds of surface plasmon modes: a weak onearound 520 nm transvers mode, and a very strong longitudinal plasmonaround 766 nm [26]. As discussed above, the longitudinal surface plasmonis crucial, where the maximum excitation of this strong surface plasmonmode with the excitation by a Raman excitation laser at 784 nm can beachieved. This ensured achieving ultimate sensitivity and very lowdetection limits.

As discussed above, the silver layer 206 is coated on the AuNR 202 inoperation 256 to form a silver coated gold nanorod (AuNR/Ag). In certainembodiments, the prepared AuNRs are covered with a thin (>1 nm) silverlayer, using the reported method [30, 34], which are incorporated byreference in their entireties. Specifically, the AuNRs formed by theoperation 252 are purified by centrifugation (10,000 rpm, 30 min) twiceto remove any excess reagents, using an ultracentrifuge (ThermoScientific, Sorvall RC6+) with the rotor F215-8X50Y. The precipitate isre-dispersed in 5 ml CTAB solution by sonication. Then, 5 ml of 1% PVPsolution and 250 μl of 0.001 M AgNO₃ are added to the AuNRs solutionwith gentle mixing. After that, 100 μl of 0.1 M ascorbic acid is addedand 200 μl of NaOH solution is added to elevate the pH to around 9, inorder to initiate the silver ion reduction reaction, such that silvercoated gold nanorods are formed.

As discussed above, the thin silver layer 106 helps maintain thelongitudinal surface plasmon wavelength as close as possible to theexcitation laser source (784 nm), in order to achieve the maximum SERSsignal. As shown by HRTEM images of (a3) to (a5) of FIG. 3A, thethickness of the silver layer 206 is about 1.7 nm. Any thick silvercoating will change the surface plasmon significantly [30]. FIG. 3(D)shows Raman signal intensity of gold nanorods, silver coated goldnanorods and nanocomposites having 4MBA according to one embodiment ofthe present invention. Specifically, FIG. 3(D) shows role of silverlayer in SERS Raman enhancing (acquisition time 10 s). As shown in FIG.3(D), the silver layer 106 enhances the SERS Raman signal of AuNR/Ag bya factor of at least 129 times compared to that of pure AuNR. Thecalculation of the enhancement factor was done by analyzing the Ramanintensity of the same peaks when the Raman molecule (4MBA) was depositedon the gold nanorods and on the AuNR/Ag nanostructures, as shown in FIG.3D.

FIG. 3(B) shows SEM and STEM images of a silver coated gold nanorodaccording to one embodiment of the present invention. As shown in FIG.3(B), (b1) is a SEM image of a AuNR/Ag, (b2) is a STEM gold EDS elementimage of the AuNR/Ag, (b3) is a STEM silver EDS layer element image ofthe AuNR/Ag, (b4) is a STEM overlapped image of the AuNR/Ag, and (b5) isa EDS cross-scanning spectra of the AuNR/Ag.

Referring back to FIG. 3(C), the silver layer epitaxial growth on thegold nanorod surface can be confirmed by the absorbance spectra. Uponthe silver layer growth, the longitudinal band of gold nanorods showed ablue shift of around 20 nm (to about 740 nm), and there were nosignificant silver band appeared in the lower wavelength, which supposesto appear in that range if the silver completely covered the goldnanorods. This result is consistent with the HRTEM images that a verythin layer of silver has formed on the gold nanorod surface. The thinsilver layer 206 helps maintain the longitudinal surface plasmonwavelength as close as possible to the excitation laser source (784 nm),in order to achieve the maximum SERS signal. Any thick silver coatingwill change the surface Plasmon significantly [30]. Further, theinserted figure in FIG. 3 (C) shows the UV-Visible spectra of four typesof nanocomposites having Anti-EpCam antibody, anti-CD44 antibody,anti-keratin 18 antibody, anti-IGF-I antibody, and a negative controlnanocomposite having anti-CD45 antibody, according to one embodiment ofthe present invention. As shown in the inserted UV-Visible spectra,after the bioconjugation of the different types of antibodies on thesurface of the silver-gold nanorod, there was still strong absorption inthe UV-Visible spectra for both longitudinal and transversal absorbance.

As discussed above, the reporter layer 210 is coated on the silver layer206 in operation 260. In certain embodiments, the reporter layer 210 maybe a Raman reporter molecule layer, and the Raman reporter molecules maybe thiolated organic molecules absorbed on the surface of the AuNR/Ag.The thiolated Raman reporter molecules are more easily assembled on thesilver surface rather than gold [38] by forming Ag—S covalent bondwithin a short period of time, for example about 3 hours, and atmoderate temperature, for example ≧45° C. In certain embodiments,different reporter molecules or marker molecules may be used. TheAuNR/Ag assembled with reporter or marker molecules can be named, M₁,M₂, M₃ . . . , M_(n), respectively, which includes the differentreporter or marker molecules, where n is a positive integer.Specifically, the silver coated gold nanorods (AuNR/Ag) obtained in theoperation 256 may be purified by centrifugation at 12,000 rpm andredispersed in deionized (DI) water. The centrifugation may be repeatedat least once to remove any excess reagents. Then thiophenol moleculescan be self-assembled on the surface of silver layer [45]. In oneexample, five thiophenol derivatives are prepared with 10 mM eachethanol based solution, in five separate conical flasks each contain 5ml of AuNR/Ag. Then 5 μl of one of 4MBA, PATP, PNTP, 4MSTP, and 4ADPS isadded and kept under stirring for about 3 hours with 45° C. to assurethat a large portion of surface attached CTAB are replaced by Raman SERSmolecules. Unabsorbed excess was removed by centrifugation once at10,000 rpm for 30 min.

As described above, in order to reduce the risks of false results thatmay raise from using single SERS nanocomposite (one Raman peak signal),multiple SERS nanocomposites can be simultaneously prepared to have aseries of discriminated peaks each corresponds to a specific SERSnanocomposite.

FIG. 4 schematically shows diagrams of preparing and using differenttypes of SERS nanocomposites for cancer cells detection according tocertain embodiments of the invention. Specifically, FIG. 4 (A) shows aschematic diagram of preparing four types of SERS nanocomposites and theRaman spectra (acquisition time 50 seconds) corresponding to each of thefour types of SERS nanocomposites according to one embodiment of thepresent invention. Referring to FIG. 4 (A), schematic diagram ofpreparing four types of SERS nanocomposites and the Raman spectra(acquisition time 50 s) corresponding to each of the four types of SERSnanocomposites are provided. Each color represents a unique AuNR/Agcovered by small organic compounds then a layer of HS-PEG-COOH and thena specific cancer cell antibody layer, where the blue color representsAuNR/Ag/4MBA/anti-EpCAM, the red color representsAuNR/Ag/PNTP/anti-IGF-1 Receptorβ, the green color representsAuNR/Ag/PATP/anti-CD44, and the magenta color-representsAuNR/Ag/4MSTP/anti-Keratin18. The nanocomposites containing 4MBA shows aspecific peak at 422 cm⁻¹, the nanocomposites containing PATP shows aspecific peak at 1372 cm⁻¹, the nanocomposites containing PNTP shows aspecific peak at 1312 cm⁻¹, and the nanocomposites containing 4MSTPshows a specific peak at 733 cm⁻¹. Thus, in one embodiment, fourdifferent SERS signals are completely separated and do not have anyoverlapping peaks.

FIG. 4 (B) shows schematically nanocomposites specifically targeting thesurface of a breast cancer cell to obtain the SERS thermal spectraaccording to one embodiment of the present invention.

FIG. 4 (C) shows schematic views that different types of SERSnanocomposites accumulate on the surface of breast cancer cells (MCF-7)and produce multi-color spectra according to one embodiment of thepresent invention.

As described above, in one embodiment, the Raman reporter molecule is atleast one of 4MBA, PATP, PNTP, 4MSTP, and other molecules with uniqueRaman spectra and intense Raman peak intensities. In one embodiment, theproduced nanocomposites include at least one of a nanocomposite having a4MBA layer, a nanocomposite having a PATP layer, a nanocomposite havinga PNTP layer, and a nanocomposite having a 4MSTP layer. All the SERRaman spectra are obtained through the detection of those Raman reportermolecules. In this way, Raman reporter molecules can be attached on thethin silver surface, while the silver coated gold nanorod still keepsthe high SERS signal enhancement. For example, when the Raman reportermolecule is 4MBA, the 4MBA attached silver coated gold rod shows sixtimes more enhancement than Raman signal in the related art.

In certain embodiments, the reporter molecules are suitable for beingdetected by methods other than SERS, and the biocompatible agent havingone or more nanocomposites are therefore configured to be applied withmethods other than SER, for specific targeting, detection, and treatmentof cancer cells or other targeted objects. In one embodiment, the reportmolecules may include one or more fluorescent agents, and the one ormore fluorescent agents may be quantum dots or fluorescent dyes.

In operation 264, the binding layer 214 is coated on the reporter layer210. In one embodiment, the binding layer 214 is a pegylated layer. Inone embodiment, the pegylated layer 214 includes a mixture of HS-PEG andHS-PEG-COOH, which serves as protective, bio-dispersive and linker tothe conjugated antibodies. Specifically, one of the precipitates fromthe previous step is redispersed in 2 ml HS-PEG-COOH (MW˜3000) solution(2 mg/ml in 2 mM NaCl), and vigorously stirred for 15 min. Then, 1.8 mlof HS-PEG solution (2 mg/ml in 2 mM NaCl) is added and kept in contactwith the SERS nanoagents at 5° C. overnight. After that, the unboundthiolated PEG is removed by centrifugation at 4000 rpm for 15 min andredispersed using probe sonication twice. The precipitate for eachcoated nanorod solution is then re-suspended in 1×PBS (phosphate buffersolution) solution.

As described above, to stabilize the prepared SERS nanocomposites, athin layer of HS-PEG(5 kD)/HS-PEG-COOH(3 kD) mixture are used, eachnanorod required around 4,200 molecules to assure complete surfacecoverage, i.e. each HS-PEG molecule requires 0.35 nm² footprint [39].This layer has to serve two purposes: first, to protect the nanorodssurface and to make the SERS nanoagents more hydrophilic and easilydisperse in aqueous medium like biological fluids, and second to providea carboxylic terminal on the surface of the SERS nanoagent, which is thelinker between the nanorod surface and the antibodies that will usedlater for targeting cancer cells. Thiolated PEG polymers are widely usedwith SERS tags and are well known as non-toxic; additionally, they donot displace Raman reporter molecules, which attach to the surface ofgold nanoparticles [40].

In operation 268, an active layer 218 is attached on the binding layer214, so as to form the nanocomposites. The active layer 218 may includeat least one of a targeting molecule and a functional molecule. Thetargeting molecule is configured to specifically bind to the target ofinterest and the functional molecule is configured to interact with thetarget of interest. In one embodiment, the targeting molecule is anantibody. The formed nanocomposites may include different antibodies,and can be named correspondingly as AB₁, AB₂, AB₃ . . . , AB_(n),respectively.

In one embodiment, a two-step conjugation assay [46] is followed to bindthe carboxylated PEG covered nanorods (SERS nanorods) with thecorresponding antibody, including an activation step and a conjugationstep.

Specifically, in the activation step, 4 ml of purified carboxylated SERSnanorods from the previous step is re-suspended in PBS buffer solutionusing sonic probe for several minutes. A mixture of NHS and EDC (0.012 geach) is added to the solution and stirred for 15 min. After that,unbound materials are washed off twice using centrifugation at 8000 rpmfor 10 min using PBS buffer.

In the conjugation step, the carboxyl-activated nanorods are redispersedin 5 ml PBS buffer solution. To each of the five prepared solutions, thecorresponding antibody is added (anti-EpCAM to AuNR/Ag/4MBA, anti-CD44to AuNR/Ag/PATP, anti-IGF-1 receptor β to AuNR/Ag/PNTP, Keratin18 toAuNR/Ag/4MSTP, and anti-CD45 to AuNR/Ag/4ADPS) and mixed thoroughly. Thereaction solution is stirred for 4 hours at room temperature. Afterthat, the antibody tagged nanorods (SERS nanoagents) are washed andre-suspended in 5 ml 1×PBS solution and kept under −20° C. for lateruse.

As described above, the formed active layer 218 may only includeantibody molecules. The active layer 218 includes antibodies that arespecifically targeting certain cancer cell surface antigens. In oneembodiment, the antibody are attached covalently with HS-PEG-COOH (—COOHterminal) and plays a role in the specific SERS nanocomposite deliveryto the cancer cells. In one embodiment, the active layer 218 includes atleast one of anti-EpCAM antibody, anti-CD44 antibody, anti-IGF-1Receptor β antibody, anti-Keratin 18 antibody, and one or moreantibodies specific to the target of interest.

In certain embodiments, the method includes attaching functionalmolecules to the binding layer 214 or the targeting molecules of theactive layer 118, to complete the active layer 218. In one embodiment,the functional molecule is a growth factor that induces certainbiological functions, including the growth, proliferation ofdifferentiation of cells or organisms. In one embodiment, the functionalmolecule is a protein, a drug, or a biological system that inducescertain biological functions, the death of cells, tissues, or organisms.The one or more drugs may be anticancer drugs, antibiotics, or antiviraldrugs. In one embodiment, the functional molecule is a virus thatspecifically disrupts the cancer cell or induces apoptosis of the cancercell. In one embodiment, the functional molecule is a plant growthfactor that improves growth of the plant.

Referring to FIG. 4 (A), schematic diagrams of preparing the four typesof SERS nanocomposites and the Raman spectra (acquisition time 50 s)corresponding to each of the four types of SERS nanocomposites areprovided. Each color represents a unique AuNR/Ag covered by smallorganic compounds then a layer of HS-PEG-COOH and then a specific cancercell antibody layer, where the blue color representsAuNR/Ag/4MBA/anti-EpCAM, the red color representsAuNR/Ag/PNTP/anti-IGF-1 Receptorβ, the green color representsAuNR/Ag/PATP/anti-CD44, and the magenta color representsAuNR/Ag/4MSTP/anti-Keratin18. The nanocomposites containing 4MBA shows aspecific peak at 422 cm⁻¹, the nanocomposites containing PATP shows aspecific peak at 1372 cm⁻¹, the nanocomposites containing PNTP shows aspecific peak at 1312 cm⁻¹, and the nanocomposites containing 4MSTPshows a specific peak at 733 cm⁻¹.

In a further aspect, the present invention is directed to a system fordetecting and monitoring at least one target by SERS. In certainembodiments, the at least one target includes cancer cells. In certainembodiments, the system includes a nanoagent, a surface-enhanced Ramanspectrometer, and a processing unit.

In certain embodiments, the nanoagent may include multiplenanocomposites prepared as described above. The multiple nanocompositesmay correspond to SERS signals of multiple colors. In certainembodiments, each nanocomposite may include a silver coated goldnanorod, a Raman reporter molecule layer assembled on the silver layer,a pegylated layer coated on the Raman reporter layer, and an antibodylayer conjugated to the pegylated layer, as described above. Thenanoagent may be applied, for example, to a blood sample or body fluidsample from a patient or a potential patient. Alternatively, thenanoagent may be applied, for example, by injection, to a patient. Dueto the specific targeting property of the antibody on the surface of thenanoagent, the nanoagent may specifically bind to, for example, one ormore cancer cells in the blood or other objects. Then an incidentradiation signal, e.g., a laser beam, may be applied to thesample/blood/object with the nanoagent, and SERS spectra are collectedusing the SERS signal from the nanoagent. The collected spectra areprocessed by the processing unit, such that the presence and/or thequantity of the one or more cancer cells or other targets can bedetermined.

In certain embodiments, the laser beam for SERS signal excitation may beone beam, or may be split in a multitude of sub-beams. In certainembodiments, the Raman spectra corresponding to the SERS agents could beintegrated in a 2D image. Moreover, the laser beam may be off-focusedsuch that the surface of analysis is increased.

In one embodiment, after necessary modification, the system may workwith other systems such as DualScan from Horiba or similar systems.

In certain embodiments, the report molecules may include one or morefluorescent agents. The one or more fluorescent agents can be quantumdots or fluorescent dyes. And the system includes an equipment that canbe used to detect the one or more fluorescent agents.

In yet another aspect, the present invention is directed to a method ofdetecting and monitoring one or more targets, such as cancer cells, bySERS, using the system as described above.

In certain embodiments, the nanocomposite in the above described systemincludes one or more functional molecules for treating the one or moretargets. In one embodiment, the functional molecule is a growth factorthat induces certain biological functions, including the growth,proliferation of differentiation of cells or organisms. In oneembodiment, the functional molecule is a protein, a virus, a phage, adrug, or a biological system that induces certain biological functions,the death of cells, tissues, or organisms. The one or more drugs may beanticancer drugs, antibiotics, or antiviral drugs.

In certain embodiments, after the treatment of the target of interest bythe functional molecules, the nanoagent provides a convenience andefficient means for monitoring the conditions of the target of interest.For example, if the target of interest is at least one cancer cell, andthe nanoagent includes functional molecules of a virus. When thenanoagent is delivered to the cancer cell by cancer cell specifictargeting molecules (such as an antibody), the virus enters the cancercell. The virus entered the cancer cell may hijack the protein synthesissystem of the cancer cell for preparing virus proteins, make multiplecopies of virus genes, and packaging new virus offspring using thosevirus proteins and genes. The cancer cell may then be destroyed by thevirus and release the virus offspring, or the cancer cell may be inducedto have an apoptosis process. The new released virus offspring's maythen infect and destroy other cancer cells that are close by oraccessible through patient's circulation system. In order to make surethe safety of the virus, the virus may be designed or engineered suchthat it only infects certain cancer cells, not normal cells.

Comparing with the structure and detection method in related art, thebiocompatible nanoagent and the method of using the nanoagent fordetecting at least one target, such as cancer cells, by SERS accordingto certain embodiments of the present invention, among other things, hasthe following advantages.

Firstly, SERS provides a high resolution, high sensitivity detectionmethod over conventional Raman method. Specifically, SERS signals aresignificant enhanced compared to the conventional Raman signal, allowingsignal collection from down to a single molecule level when variousnoble metals of rough surfaces or nanomaterials are used. Theenhancement factors for the Raman-scattering signals of SERS can be morethan one million-fold compared with normal Raman signals. Therefore,SERS has a significant potential to be used in bio-medical applications[9].

Secondly, in certain embodiments of the present invention, silver coatedgold nanorods (AuNR/Ag) are used to prepare the nanoagent for SERSdetection. The AuNR/Ag shows stronger spectroscopic properties comparedto AuNR [31] or silver nanoparticles. In one embodiment, metallicnano-silver does not suppress surface plasmons as strongly as nano-gold[31]. Further, the silver-gold interface (AuNR/Ag) core-shells have40-50% more light scattering capacity compared to pure AuNRs [30], whichmakes them excellent candidates for SERS bio-medical applications wheresingle molecule level detection limits are required. Accordingly, incertain embodiment, the AuNR/Ag in the nanoagent is superior to goldnanoparticles or silver nanoparticles.

Thirdly, in certain embodiments of the present invention, four differentantibodies against specific surface antigens of breast cancer cell lineMCF-7 are used for preparing the biocompatible nanocomposite for SERSdetection. The four antibodies include Anti-EpCam antibody, anti-CD44antibody, anti-keratin 18 antibody, and anti-IGF-I antibody. The fourcorresponding antigens are highly expressed in certain cancer cells,especially breast cancer cells. The four types of antibodies andcorresponding SERS reporter molecules can be represented by differentcolors in SERS detection. When the SERS signals represented by differentcolors are combined, the multicolor combination shows high sensitivityand accuracy than single color detection, and prevents signaloverlapping.

In yet another aspect, the present inventions relates to ananocomposite. In one embodiment, the nanocomposite includes ananostructure and an active layer conjugated to the nanostructure.

In certain embodiments, the nanostructure has a spherical shape, atubular shape, a cylindrical shape, or a rod-like triangular shape. Incertain embodiments, the nanostructure is formed by at least onenanomaterial. In one embodiment, the nanostructure includes beplasmonically active nanostructures of gold, silver, gold/silver,copper, iron, etc. In one embodiment, the nanostructure includes carbonnanotubes and small graphene. In one embodiment, the nanostructureincludes oxide structure of Fe_(x)O_(y), TiO₂, SiO₂. In certainembodiments, the nanomaterial may be quantum dots, nanorods, nanowires,nanofiber, fullerene, and the like. These nanostructures may provideRaman signal through the linkage of an organic molecule, such asattaching Raman reporter molecules as described above, or/and by theirown structure, such as carbon nanotubes/graphenes.

In certain embodiments, the active layer includes functional moleculethat is configured to interact with a target of interest. In oneembodiment, the target of interest includes a cancer cell, a pathogen,or a plant. In one embodiment, the functional molecule includes a virus,a phage, a drug, a growth factor, antibiotics, a gene, a plasmid, avaccine, a plant growth agent, an anti-fungal, a fertilizer, herbicides,an antibody that specifically binds to S100 calcium-binding protein B(S100B), or other biological active molecules.

In certain embodiments, the herbicides include several different groupsor types of herbicides that are suitable for being attached to thenanostructure of the present invention. One group of herbicides isacetolactate synthase (ALS) inhibitors. The ALS inhibitors include, forexample, (imidiazolines) imazamox,(sulfonylaminocarbonyltriazolinones)propoxycarbazone, (sulfonylureas)halosulfuron, imazapic, imazapyr, imazethapyr, mesosulfuron,primisulfuron, sulfometuron, thifensulfuron, and triflusulfuron. Anothergroup of herbicides is microtubule assembly inhibitors, which includes,for example, (dinitroanalines) oryzalin. A further group of herbicidesis synthetic auxins, which includes, for example, (benzoic acid)dicamba, (phenoxyacetic acids) MCP, (pyridines) aminopyralid, (quinolinecarboxylic acids) quinclorac, clopyralid, fluroxypyr, mecoprop (MCPP),picloram, and triclopyr.

The functional molecules may be linked to the nanostructure by variousapproaches. In certain embodiments, by mixing the nanostructure and thefunctional molecules, the functional molecules are linked to thenanostructure through physisorption/chemisorption, pi-pi stacking (forcarbon nanotubes and graphenes), covalent bonding, hydrogen bonding,etc. In certain embodiments, an intermediary layer is used forconnecting the functional molecules to the nanostructure. Theintermediary layer may be at least one of a pegylated layer as describedabove, a polyethyleneimine (PEI) layer, a polymer layer, or any otherlayer that provide chemical functionalities for the covalent attachment.

Examples Example 1 Preparing a Biocompatible Nanoagent

In all preparation procedures, deionized water (DI water, 18 Ω/cm) wasused. The following chemicals were purchased from Sigma-Aldrich and usedwithout further purification: Gold (III) chloride trihydrate (99%),sodium borohydride (99%), L-ascorbic acid (98%), 4-mercaptobenzoic acid(4MBA), p-aminothiophenol (PATP), p-nitrothiophenol (PNTP),4-methylsulfanylthiophenol (4MSTP), and 4-aminophenyldisulfide (4APDS),Polyvinylpyrrolidone (PVP) (MW˜10,000), N-hydroxysuccinimide (NHS),1N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).Silver nitrate was purchased from Fisher Scientific.Hexadecyltrimethylammoniumbromide (CTAB 99%) was purchased from MPBiomedicals. SH-PEG (Mw˜5000) was purchased from Nanocs (95%).HS-PEG-COOH (Mw˜3000) was purchased from Sigma-Aldrich.

All antibodies used (anti-EpCAM, anti-CD44, anti-CD45, anti-IGF-1Receptor β, anti-cytokeratin18) were purchased from Cell Signaling athigh purity.

Human breast carcinoma cell line (MCF7) and fibroblast normal skin cellline were purchased from the American Type Culture Collection (ATCC).Culture media, including culture supplies, were purchased from FisherScientific.

In Example 1, the nan-agent according to certain embodiments of thepresent application was synthesized as follows.

Synthesis of gold nanorods: gold nanorods (AuNRs) with turned size wereprepared according to the seed mediated method by Nikoobakht [26].Briefly, the seed solution was first prepared by mixing 5 ml of 0.2 MCTAB solution with 5 ml of 0.0005 M HAuCl₄, and then adding 600 μl ofNaBH₄ with stirring for two minutes. To synthesize AuNRs with an aspectratio around 3, 5 ml of 0.2 M CTAB was mixed with 150 μl of 0.004 Msilver nitrate solution, then 5 ml of 0.001 M HAuCl₄ was added andmixed, after that 70 μl of 0.0788 M ascorbic acid was mixed with thesolution and finally 12 μl of seed solution was added. The mixedsolution was kept at 30° C. for 40 minutes without any further stirring.

Coating of AuNR with a thin shell silver layer to form a silver coatedgold nanorod (AuNR/Ag): the prepared AuNRs were covered with a thin (>1nm) silver layer, using the previously reported method [30, 44]. AuNRsfrom the synthesis step were purified by centrifugation (10,000 rpm, 30min) twice to remove any excess reagents, the precipitate wasre-dispersed in 5 ml CTAB solution by sonication, then 5 ml of 1% PVPsolution and 250 μl of 0.001 M AgNO₃ were added to AuNRs solution withgentle mixing. After that 100 μl of 0.1 M ascorbic acid was added and200 μl of NaOH solution was added to elevate the pH to around 9, inorder to initiate the silver ion reduction reaction.

Assembling SERS compounds on the surface of the AuNR/Ag: the AuNR/Agfrom the previous step were purified by centrifugation at 12,000 rpm andredispersed in deionized water, twice to remove any excess reagents.Thiophenol molecules can be self-assembled on the surface of the silverlayer [45]. Five thiophenol derivatives were prepared with 10 mM eachethanol based solution, in five separate conical flasks each contain 5ml of AuNR/Ag. 5 μl of 4MBA, PATP, PNTP, 4MSTP, or 4ADPS were addedseparately and kept under stirring for 3 hours at 45° C., this stepassured that a large portion of surface attached CTAB were replaced byRaman SERS molecules. Unabsorbed excess was removed by centrifugationonce at 10,000 rpm for 30 min.

Coating with HS-PEG and HS-PEG-COOH: each precipitate from the previousstep was redispersed in 2 ml HS-PEG-COOH (MW˜3000, 2 mg/ml in 2 mM NaCl)solution and vigorously stirred for 15 min, then 1.8 ml of HS-PEG (2mg/ml in 2 mM NaCl) solution was added and kept in contact with the SERSnanoagents at 5° C. overnight. After that, the unbound thiolated PEG wasremoved by centrifugation at 4,000 rpm for 15 min and redispersed usingprobe sonication twice. The precipitate for each coated nanorod solutionwas re-suspended in 1×PBS (phosphate buffer solution) solution.

Conjugation of coated SERS nanorods with antibodies: a two-stepconjugation assay [46] was followed to bind the carboxylated PEG coverednanorods (SERS nanorods) with the corresponding antibody.

Activation Step:

4 ml of purified carboxylated SERS nanorods from the previous step wasre-suspended in PBS buffer solution using sonic probe for severalminutes. A mixture of NHS and EDC (0.012 g each) was added to thesolution and stirred for 15 min. After that, unbound materials werewashed off twice using centrifugation at 8,000 rpm for 10 min and PBSbuffer.

Conjugation Step:

the carboxyl-activated nanorods were redispersed in 5 ml PBS buffersolution. To each of the five prepared solutions, the correspondingantibody was added (anti-EpCAM to AuNR/Ag/4MBA, anti-CD44 toAuNR/Ag/PATP, anti-IGF-1 receptor (3 to AuNR/Ag/PNTP, Keratin18 toAuNR/Ag/4MSTP, and anti-CD45 to AuNR/Ag/4ADPS) and mixed thoroughly. Thereaction solution was stirred at for 4 hours at room temperature. Afterthat, the antibody tagged nanorods (SERS nanoagents) were washed andre-suspended in 5 ml 1×PBS solution and kept under −20° C. for lateruse.

Characterization of the Nanoagent Prepared from Example 1

The nanoagents prepared from Example 1 were characterized by variety ofmethod, such as scanning electron microscopy (SEM), transmissionelectron microscopy (TEM), high resolution TEM (HRTEM), SERS, UV-Visspectroscopy.

For TEM characterization, the morphology and size of the gold nanorods(AuNRs) according to Example 1 were determined by TEM, JEM-2100F (JEOLUSA, Peabody, Mass., USA) with an accelerating voltage of 80 kV. Highresolution TEM (HRTEM) imaging was performed at 200 kV. A few drops eachof samples suspended in water were deposited on holey-carbon coatedcopper grids, which were then allowed to dry for 15 minutes on filterpapers. The average rod size and the size distribution of each samplewere determined by using Image J image analysis tool. The PEG-coatedgold nanorod and the protein coated gold nanorod samples were positivelystained with 2% uranyl acetate dissolved in 70% ethanol in order toenhance the protein coating layer around the AuNR. The TEM was alsoequipped with an EDAX Genesis energy dispersive spectroscopy (EDS) ofX-ray detection system. Combined with Scanning Transmission ElectronMicroscopy (STEM), elemental mapping of nanorods can be performed withclose to 1 nm lateral resolution Annular dark field (ADF) imaging underSTEM mode was performed with 1.5 nm spot size and 20 cm camera lengthwith a JEOL dark field detector.

For SEM characterization, MCF-7 cells were grown on Thermanox® plasticcoverslip (NUNC, Rochester, N.Y.) for 24 hours. Samples were treatedwith SERS nanoagents for 30 minutes. MCF-7 cells were fixed primarilywith 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, followed by asecondary fixative of 2% OsO4 in 0.1 M phosphate buffer. All of thesamples were washed thoroughly with 0.1 M phosphate buffer, dehydratedwith ascending percentages of ethanol solution, and then dried usingHexamethyldisilazane (HMDS) reagent (EMS, Hatfield, Pa.). Each driedsample was coated with a thin film of carbon (˜3 nm) and visualizedunder a SEM, JEOL JSM-7000F (JEOL USA, Peabody, Mass.) also equippedwith an EDAX EDS detection system with an accelerating voltage of 15 kVand a working distance of ˜10 mm.

Referring to the HRTEM images as shown in (a1) and (a2) of FIG. 3 (A),the particle length and diameter of the AuNRs are approximately 36±0.80nm and 12±0.41 nm, respectively. Referring to the HRTEM images as shownin (a1) and (a2) of FIG. 3 (A), the silver film has a thickness of about1.7 nm.

FIG. 3 (B) shows images of AuNR/Ag, where (b1) is a SEM image, (b2) is aSTEM gold EDS element image, (b3) is a STEM silver EDS layer elementimage, (b4) is a STEM overlapped image, and (b5) is a EDS cross-scanningspectra. The result indicates that the silver atoms give the highesttendency for the outside layer arrangement in this bimetal composition.The thin silver layer helps maintain the longitudinal surface plasmonwavelength as close as possible to the excitation laser source (784 nm),in order to achieve the maximum SERS signal.

FIG. 5A and FIG. 5B show visualization of SERS nanoparticles on thecells' surface, where FIG. 5A shows SEM images with EDS elementalanalysis of nanocomposites on the MCF-7 cell surface according to oneembodiment of the present invention, and FIG. 5B shows TEM images ofnanocomposites cluster on the surface of an MCF-7 cell according to oneembodiment of the present invention. As shown in FIG. 5A, (a1) is a SEMimage of the nanorods cluster on a MCF7 cell surface, (a2) is an imageshowing Au EDS elemental analysis of the nanorods cluster, and (a3) isan merged image of the nanorods cluster. As shown in FIG. 5B, (b1),(b2), and (b3) are TEM images of the nanorods (SERS nanoagents) clusteron the surface of an MCF7 cell with different magnification, showing howthe nanorods accumulate on the surface of an MCF7 cell. FIGS. 5A and 5Bclearly show that the SERS nanoagents accumulated on the surface of thecells.

UV Visible Spectra: 100 μg/ml solution of AuNRs, AuNR/Ag, and all SERSnanoagents were scanned from 400-900 nm using Shimadzu (UV-Visible-NIR)spectrophotometer. The data was re-constructed using software.

FIG. 3 (C) shows UV-Vis spectra of AuNR and AuNR/Ag, and the insertedimage shows UV-Vis spectra of SERS nanoagents. As shown in FIG. 3 (C),the two dimensions, particle length and diameter of the AuNRs andAuNR/Ag, are adequate to form two kinds of surface plasmon modes: a weakone around 520 nm transvers mode, and a very strong longitudinal plasmonaround 766 nm [26]. The longitudinal surface plasmon is crucial, and themaximum excitation of this strong surface plasmon mode can be achievedwhen excited by a Raman excitation laser at about 784 nm. This ensuresultimate sensitivity and very low detection limits when uses SERS forcancer cell detection.

Further, the silver layer epitaxial growth on the gold nanorod surfacecan be confirmed by the absorbance spectra. Upon the silver layergrowth, the longitudinal band of gold nanorods showed a blue shift ofaround 20 nm (to about 740 nm), and there were no significant silverband appeared in the lower wavelength, which supposes to appear in thatrange if the silver completely covered the gold nanorods. This result isconsistent with the HRTEM images that a very thin layer of silver hasformed on the gold nanorod surface.

As shown in the inserted image of FIG. 3 (C), after the bioconjugationof the different types of antibodies on the surface of the silver coatedgold nanorod, there were still strong absorption in the UV-Vis spectrafor both longitudinal and transversal absorbance.

To evaluate the ability of anti-EpCam, CD44, Keratin 18, and IGF-Iantibodies to specifically target cancer cells, as well as todiscriminate between the two cell lines (MCF-7 and fibroblast),immunocytochemistry techniques were conducted. The antibody binding wasidentified by the use of a secondary antibody labeling method with fourdifferent colors.

Breast adenocarcinoma (MCF-7 cell line), and normal skin fibroblast(BJ-1 cell line) were co-cultured in two-well chamber slides in adensity of 15⁴ cells/well with the percentages of 90% fibroblastic BJ-1cells and 10% cancerous MCF-7 cells. The mixed cells were then incubatedfor 24 hours for attachment. Post incubation, the cells were washed with1× of phosphate buffer saline solution 3×5 min each. 200 μl of highpurity methanol was added to each well and incubated for 20 min at roomtemperature for fixation. The methanol was removed, and the cells werewashed with 1× of phosphate buffer saline solution 3×5 min each.Subsequently, 200 μl of blocking buffer containing (1×PBS/5% BSA) wasadded to each well and incubated for 30 min at room temperature. In themeantime, a 1:200 dilution of each antibody-Anti-EpCam, Anti-CD44,Anti-Keratin 18, and Anti IGF-I (Cell Signaling Technology, Boston,Mass.)—was prepared using blocking buffer in separate labeled tubes. Theblocking buffer was removed, and the diluted antibodies were added toeach well and incubated over night at 4° C. The cells were then washedwith 1× of phosphate buffer saline solution for 3×5 min each. Fourdifferent goat polyclonal secondary antibodies to mouse IgG withdifferent fluorescent properties—Alexa Flour 488, 555, 594, and 647(abcam, Cambridge, Mass.)—were used in a dilution of 1:500 with blockingbuffer. The secondary antibodies were added to each well and incubatedfor 1 h in the dark at room temperature. The cells were washed with 1×of phosphate buffer saline solution 3×5 min each, and then 200 μl of0.5% μg/ml of 4′,6-diamidino-2-phenylindole (DAPI) was added andincubated for 5 min at room temperature in the dark to perform nuclearstaining. The cells were mounted with mounting medium, covered with thecover slip, and the edges were sealed. Finally, the cells were kept inthe dark at 4° C. until examination under the fluorescence microscopeusing an Olympus BX 51 microscope.

FIG. 6 schematically shows the immunocytochemistry staining (ICC) ofMCF-7 cells in mixed culture with fibroblast cells BJ-1 according to oneembodiment of the present invention. As shown in FIG. 6, the top rowshows ICC of cells with anti-EpCAM, anti-CD44, anti-Keratin 18, andanti-IGF antibodies (1:200), respectively, followed by (1:500) goatanti-mouse IgG (Alexa fluor 555, 594, 488, and 647). The middle rowshows DAPI nuclear staining of both cell lines. The bottom row showsmerged images. The white arrows indicate the presence of antigenslocalized on the surface of breast cancer cells, and not on the surfaceof fibroblast cells.

As presented in FIG. 6, the anti-EpCAM antibody, anti-CD44, anti-Keratin18 and anti-IGF-I were found to selectively bind MCF-7 cancer cells andnot to the normal fibroblast cells. As a result, this method couldfacilitate the specific delivery of SERS nanoagents to breast cancercells only.

SERS: Referring back to FIG. 3 (D), the silver layer enhances the Ramansignal by a factor 129 times (SERS) compared to that of pure AuNR.

Example 2 Using the Prepared Nanoagent for Cancer Cell Detection by SERS

Breast adenocarcinoma (MCF-7 cell line) and skin fibroblast (BJ-1 cells)were purchased from American Type Culture Collection (ATCC). Both celllines were primarily cultured in 75 cm² tissue culture flasks andsupplemented with the recommended medium—Dulbecco minimum essentialmedium (DMEM), and Eagle's minimum essential medium (EMEM) respectively,both containing 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin (PS), the cells were incubated at 37° C. in humidifiedincubator and 5% CO₂. The medium was changed every 48 h with freshmedium until confluent.

For SERS analysis, mixture of cancerous MCF7 cell line and BJ-1 cellline were seeded in 4 well chamber slides in a density of 10⁵ cells/welland a percentage of 90% BJ-1 cells, 10% MCF-7 cells. The cells weresupplemented with complete growth medium and incubated overnight forattachment. After incubation, the medium was interchanged with normalgrowth medium supplemented by 40 μg/ml of SERS nanoagents (10 μg of eachSERS nanoagents) and the cells were further incubated for 30 min at 37°C. The cells were washed for 5 minutes 3 times with 1×PBS, and 2%formaldehyde was added for fixation. After 20 min, the cells were washed6 times (3 times with 1×PBS, and 3 times with DI water). The cells wereleft to dry and stored at −20° C. Untreated cells were used as anegative control.

SERS images were collected from the samples using Confocal Ramanspectrometer (Horiba Jobin Yvon LabRam HR800, Edison, N.J.) assembledwith He—Ne laser (784 nm) and three Olympus BX-51 lenses with 100×micro-objectives magnitude connected to a Peltier-cooled CCD camera. Thespectra were collected using 600-line/mm gratings with the sameacquisition time. The spectrometer also has a three-dimensional (3-D)(x-y-z) automatic adjustable stage that can map Raman scanning for aspecific area at a minimum distance of 1 μm. In all measurements, theRaman spectrometer was calibrated using the Si—Si Raman signal, which islocated at a 521-cm⁻¹ Raman shift.

FIG. 7A shows Raman mapping images of cells according to embodiments ofthe present invention, where (a1) shows Raman mapping images fortargeting a single MCF-7 cancer cell among fibroblast cells with fourdifferent SERS nanoagents; (a2) shows Raman mapping images of a cancercell without using any SERS nanoagents; and (a3) shows Raman mappingimages of a fibroblast cell (normal cell) with four SERS nanoagents. Asshown in FIG. 7A, normal cells as shown in (a3) and MCF-7 cells withoutSERS nanoagent have not reveal any Raman signal in scanned regions.These Raman mapping images have clearly confirmed that multiple SERSnanocomposites (blue, red, green, and magenta referred to 4MBA, PNTP,PATP, 4MSTP respectively) of a nanoagent are simultaneously targetingthe same MCF-7 cell within 30 minutes of incubation with 8 s Ramanacquisition time.

In order to detect the cancer cell MCF-7 in real blood or separatedwhite blood cells, a few of cancer cells (5, 50, 500 or 5000 cells)spiked with (100 μL) whole blood or the separated white blood cellsolution. In one embodiment, the two samples (separated blood and wholeblood) contain about 50 cancer cells in 7 million white blood cells.Then 50 μg/ml of SERS nanoagents mixture were added to incubate for 30minutes.

FIG. 7B shows Raman mapping images of cells according to certainembodiments of the present invention, where (b1) shows Raman mappingimages of a single cancer cell, MCF-7, among millions of white bloodcells, using a nanoagent having four types of nanocomposites; (b2) showsRaman mapping images of a single cancer cell, MCF-7, among millions ofwhole blood cells, using a nanoagent having four types ofnanocomposites; and (b3) shows SERS mapping signal collected from thewhite blood cells only, i.e., without presence of cancer cells, using ananoagent having four types of nanocomposites. As shown in (b1) and (b2)of FIG. 7B, four different colors show the four different SERSnanoagents were detected in the same cell which were located in thedifferent place of the cancer cell surface and some were overlapped eachother. However, signals from the four types of SERS nanocomposites canbe distinguished because of the colors, which means the specific Ramanpeaks. As shown in (b3) of FIG. 7B, there is no specific Raman signalfrom the white blood cells.

In order to estimate how fast SERS signal could be detected, an MCF7cell was targeted and scanned several times with different acquisitiontime (1 s, 2 s, 3 s, 4 s, 5 s). FIG. 7C shows SERS signal collected withdifferent time periods from 1 second(s) to 5 s, using a nanoagent havingfour types of nanocomposites according to one embodiment of the presentinvention. As shown in FIG. 7C, SERS signal could be significantlydetected mostly within 3 s. The intensity of the Raman signal can bedetected variable from 1-8 s depended on the different antibodies of thenanocomposites. The SERS nanoagents having the multiple nanocompositeswere successfully targeted on the MCF-7 single cell and detected byRaman among millions of fibroblast cells.

FIG. 7D schematically shows SERS linear scanning position and SERSsignal of a selected single cancer cell according to one embodiment ofthe present invention, where (d1) shows SERS linear scanning position ofa selected single cancer cell, and (d2) shows SERS signal of a selectedsingle cancer cell scanned linearly four times along the line in FIG. 7D(d1), each time with a specific scanning range corresponding to one ofthe four types of SERS nanocomposites. As shown in FIG. 7D, a singlecancer cell was selected and scanned linearly four times, each time witha specific scanning range corresponding to that specific SERS nanoagent.FIG. 7D shows that SERS signals come from only the MCF-7 cell and thereis no significant signal from the blood cells.

In order to ensure that SERS nanoagent having multiple type ofnanocomposites were selectively targeting MCF-7 cells because they aredirected to these cells by the antibodies, a negative SERS nanoagent wasprepared and tested. Anti-CD45 is a biomolecule known to target whiteblood cells rather than the MCF-7 cells. The negative SERS nanocompositeof AuNR/Ag/4ADPS/CD45 was prepared and incubated with the same sample ofmixed white blood cells and few MCF-7. SERS images were then collectedto see if any signal from the cancer cells was received. The resultswere in complete agreement with our hypothesis, where the SERS signalwas obtained from the white blood cells and not from the cancer cells.

FIG. 8 shows images of SERS signal of a mixture of MCF-7 cell and whiteblood cells, using the negative nanocomposite having CD45, according tocertain embodiments of the present invention. As shown in (a1) to (a3)of FIG. 8, there was no SERS signal from the MCF-7 cell and all SERSsignal coming from the white blood cells, when using the negativenanoagents having CD45.

In summary, according to certain embodiment of the present invention, itis able to detect SERS signals from a single cancer cell among millionsof blood cells in a short period of time. The nanostructural agents,formed of four types of silver decorated gold nanorods, were designed tohave high optical absorption in the near-infrared region (NIR) and tomatch the emission of the laser excitation. All four SERS nano-reporterswere used for the multicolor superimposed identification of the cancercells. Each agent, with unique spectral features, was assigned adifferent color and the identification of the individual cancer cells inblood was performed based on the overlapping of four colors in a2-dimensional scanning environment. SERS using those nanoagent cansuccessfully detect and image with high resolution a low population ofbreast cancer cells in peripheral blood or separated white blood cells(for example, 1 in 1 million). Such a multi-spectroscopic approachoffers the opportunities of accurate and high sensitivity detection ofsingle cancer cell in blood, given narrow SERS bandwidths of thenano-reporter spectra. Thus, certain embodiments of the presentinvention provide ultrafast and high specificity detection of the earlyclinical detection of a multitude of cancer cells or various pathogensin blood.

In the past, CTCs were thought to spread only during the final stages ofmalignant progression [41, 42]. However, recent researches havedemonstrated that CTCs also can be found in the bloodstream during earlystages [43]. Consequently, the fast detection of CTCs has become majorimpact factor for treatment and providing information about theaggressiveness of a tumor, how well patients are responding totreatment, and why some patients do not respond to a specific therapy.Certain methods usually required an enrichment and separation steps ofcancerous cells from normal cell which is tedious and time consumingprocedures [41]. However, comparing with related art, the presentinvention, for the first time, is able to detect a single cancer cell(CTC) quickly and within 1 to 7 million of blood erythrocytes cellswithout any enrichment assay.

In certain embodiments, the nanoagents of the present disclosure mayalso be used to treat the target of interest by the functionalmolecules, and monitor the change of the target of interest.

Example 3 Attachment of an Anticancer Drug-Doxorubicin to theNanostructure to Form the Nanocomposite of the Present Invention

In certain embodiments, one type of the nanocomposites is formed withthe functional molecule of anticancer drug of doxorubicin (DOX). FIG. 9Ashows incorporation of the anticancer drug doxorubicin to ananostructure according to certain embodiments of the present invention.As shown in FIG. 9A, a nanostructure before adding the anticancer drughas a —COOH group disposed on the outer surface of the nanostructure.The nanostructure includes a gold nanorod, a silver layer surroundingthe gold nanorod, a Raman reporter molecule layer coated on the silverlayer (in this example, the Raman report molecule is RATP. In otherexample the Raman molecule may also be 4MBA, PNTP, 4MSTP, etc.), and apegylated layer coated on the Raman reporter layer. The pegylayted layerprovides the —COOH groups. As shown in FIG. 9A, the nanostructure havingthe —COOH groups on the outer surface is mixed with the DOX, andanti-EpCAM is also added, such that both the DOX and the anti-EpCAM maybe bonded or attached to the —COOH groups of the pegylated layerrespectively, to form the nanocomosite of NS-DOX-EpCAM, wherein NSrepresent the nanostructure, DOX represents the anticancer drugdoxorubicin, and EpCAM represents the anti-epithelial cell adhesionmolecule antibody.

FIG. 9B shows UV-Visible spectra of the nanocomposite of FIG. 9Acollected using a Shimadzu UV-Vis NIR optical absorption spectrometer.As shown in FIG. 9A, the spectrum of the AuNR/Ag/PATP/PEG-DOX-EpCAMincludes multiple specific peaks below 300 nm and a strong peak at alittle above 500 nm, corresponding to the spectrum of DOX, whichindicates that DOX is attached to the nanostructure efficiently with thehelp of the pegylated layer.

Example 4 Attachment of Functional Molecules to the Nanostructure toForm a Nanocomposite for Enhancing the Bio-Activity of BiologicalSystems

In certain embodiments, functional molecules may be attached tonanostructures to form nanocomposites. The nanocomposites, when beingdelivered to the plant or a biological system, are able to enhancingactivities of the plant or the biological system.

The nanostructure may have a spherical shape, a tubular shape, acylindrical shape, or a rod-like triangular shape. The nanostructure maybe formed from nanomaterials of gold, silver, gold/silver, copper, iron,Fe_(x)O_(y)TiO₂, SiO₂, and carbon.

The functional molecules include plant hormones. Suitable plant hormonesmay include auxins, cytokinins, gibberellins, and abscisic acids. Oneexample of the auxin is indol-3-ylacetic acid. Examples of the cytokinininclude kinetin and zeatin. One example of the gibberellins isgibberellic acid.

The functional molecules include herbicides. As shown in FIG. 10A, ananostructure having a —COOH group on the outer surface is provided. Thenanostructure has a gold nanorod, a silver layer coated on the outersurface of the gold nanorod, and a pegylated layer attached to the outersurface of the silver layer. The pegylated layer provides the —COOHgroups. As shown in FIG. 10A, the nanostructure having the —COOH groupson the outer surface is mixed with the herbicides picloram to form thenanocomposite NS—PI, where NS represents the nanostructure as describedabove, and the PI represents the herbicides picoram.

FIG. 10B shows UV-Visible spectra of the nanocomposite of FIG. 10A. Asshown in FIG. 10A, the spectrum of the AuNR—PI includes a peak at alittle less than 300 nm, corresponding to the spectrum of PI, whichindicates that PI is attached to the nanostructure efficiently with thehelp of the pegylated layer.

In another example, as shown in 10C, the herbicides dicamba (DI) may beattached to —NH₂ surface groups of a nanostructure to form thenanocomposite NS-DI, where NS represents the nanostructure having —NH₂groups on the outer surface of the nanostructure, and DI represents theherbicide dicamba.

In another example, as shown in 10D, the plant hormones 3-indolylaceticacid (3IAA) may be attached to —NH₂ surface groups of a nanostructure toform the nanocomposite NS-3IAA, where NS represents the nanostructurehaving —NH₂ groups on the outer surface of the nanostructure, and 3IAArepresents the hormone 3-indolylacetic acid.

In another example, as shown in 10E, the plant hormones gibberellic acid(GA) may be attached to —NH₂ surface groups of a nanostructure to formthe nanocomposite NSR-GA, where NS represents the nanostructure having—NH₂ groups on the outer surface of the nanostructure, and GA representsthe hormone gibberellic acid.

Example 5 Attachment of Drug Gambogic Acid (GA) to Single-Walled CarbonNanotube (SWCNT) and Graphene (Gn) for Treating Cancers

In this example, the nanostructure includes SWCNT and Gn, and the drugfor cancer treatment is GA. The description of the nanocomposite can befound in Saeed, et al, J. Applied Toxicology, 2014, 34: 1188-1199, whichis incorporated herein by reference in its entirety.

In this example, Gn and SWCNT were used to deliver the naturallow-toxicity drug GA to breast and pancreatic cancer cells in vitro, andthe effectiveness of this complex in suppressing cellular integrity wasassessed. Cytotoxicity was assessed by measuring lactate dehydrogenaserelease, mitochondria dehydrogenase activity, mitochondrial membranedepolarization, DNA fragmentation, intracellular lipid content, andmembrane permeability/caspase activity. The nanomaterials showed notoxicity at the concentrations used, and the antiproliferative effectsof GA were significantly enhanced by nanodelivery. The results suggestthat these complexes inhibit human breast and pancreatic cancer cellsgrown in vitro. This analysis represents a first step toward assessingtheir effectiveness in more complex, targeted, nanodelivery systems.

Gn was purchased from Gaia Chemical Corporation (New Milford, Conn.,USA). Gn structures were synthesized on Fe/Mo/MgO (1:0.1:110 molarratio). The catalyst system was prepared as previously reported(Dervishi et al., 2012). The graphitic nanomaterials were synthesizedusing an RF generator in an argon methane environment. The as-producedGn nanostructures were purified with hydrochloric acid. For betterdispersion in an aqueous solution, the nanosized, few-layer thick Gnsamples were functionalized with carboxylic functional groups. Eachsolution was continuously washed with distilled water and finally driedin an oven at 100° C. for 12 h.

Gn and SWCNT (10 μg/ml) were mixed with GA (0.5, 0.75 and 1 μg/ml) to bedescribed as GA+Gn and GA+SWCNT. First, the samples were vortexed at ahigh speed for at least 4 h to allow the drug to bind to thenanomaterials via π-π stacking. The samples were then centrifuged andwashed three times with distilled water to remove the unbound drug. Theloading concentrations of the GA on Gn and SWCNT were determined byUV-Vis absorbance at 360 nm measured using a Shimadzu UV-3600spectrophotometer (Shimadzu Scientific Instruments, Colombia, Me., USA).

TEM measurements of the carbon nanomaterials indicated that the SWCNThad a diameter of 1.5-2 nm, and the few-layer Gn structures had anaverage size of 100-200 nm. GA was loaded onto the surface of Gn andSWCNT, and UV-Vis analysis of GA+Gn and GA+SWCNT showed a peak at 360nm, which is the characteristic wavelength for the GA structure. Theamount of drug loaded was calculated by peak absorbance at 360 nm aftersubtracting the background of absorbance from Gn and SWCNT. It isdetermined that, after vortexing, 98% of the free GA was bound to the Gn(GA−Gn=0.98: 10 μg/ml) and that 88% was bound to the SWCNT(GA−SWCNT=0.88: 10 μg/ml). Although there was a modest toxicity level athigh concentrations from both nanomaterials as described above in bothcell lines MCF-7 and Panc-1, there was no significant toxicity measuredat 10 μg/ml.

The possible use of GA for the treatment of cancer is a growing area ofstudy, and, while this naturally derived drug has shown promise, its useis limited by its poor solubility and inadequate oral bioavailability.This example reports the first use of carbon-based nanomaterials, Gn andSWCNT, for the efficient delivery of GA to cancer cells, such as breastcancer cells and pancreatic cancer cells.

Example 6 Attachment of Drug Parthenolide (PTL) to FunctionalizedNanographene (fGn) for Treating Cancers

In this example, the nanostructure includes fGn, and the drug for cancertreatment is PTL. The description of the nanocomposite can be found inKarmakar et al, RSC Adv., 2015, 5: 2411-2420, which is incorporatedherein by reference in its entirety.

The naturally derived compound, parthenolide (PTL), is known for itsanti-inflammatory and anticancer activity, but its poor water solubilitylimits its clinical value. In this example, carboxyl-functionalizednanographene (fGn) delivery is used to overcome the extremehydrophobicity of this drug. A water-soluble PTL analog,dimethylaminoparthenolide (DMAPT), was also examined for comparison withthe anticancer efficacy of our PTL-fGn complex. Delivery by fGn wasfound to increase the anticancer/apoptotic effects of PTL (but notDMAPT) when delivered to the human pancreatic cancer cell line Panc-1.The IC50 value for PTL decreased from 39 mM to 9.5 mM when delivered asa mixture with fGn. The IC50 of DMAPT did not decrease when delivered asDMAPT-fGn and was significantly higher than that for PTL-fGn. There weresignificant increases in reactive oxygen species (ROS) formation and inmitochondrial membrane disruption in Panc-1 cells after PTL-fGntreatment as compared to PTL treatment, alone. Increases in toxicitywere also seen with apoptosis detection assays using flow cytometry,ethidium bromide/acridine orange/DAPI staining, and TUNEL. Thus, fGndelivery was successfully used to overcome the poor water solubility ofPTL, providing a strategy for improving the effectiveness of thisanticancer agent.

In this example, Gn was purchased from Angstron Materials Inc. (Dayton,Ohio, USA). Gn (10 mg) was added to a mixture of H25O4 and HNO3. Themixture was sonicated for 4-5 h and then filtered through a 0.2 mm GTTPmembrane (Millipore, USA) and washed with deionized water several times.The resulting fGn powder was oven-dried overnight at 100° C. and thenplaced in a vacuum desiccator. For use in subsequent experiments, thefGn powder was re-dispersed in deionized water by sonication. PTL wasobtained from Sigma-Aldrich (USA). DMAPT was synthesized from PTL aspreviously reported and was used as the fumarate salt. PTL and DMAPTwere dissolved in DMSO to create 10 mM stock solutions. To create thePTL-fGn and DMAPT-fGn complexes, the drug was added to fGn solution inmedia (final drug-fGn complex concentrations were 1, 10, 20, 30, 50, and100 mM drug and 10 mg/ml fGn), sonicated for 1 h, and then stored at 4°C. before being incubated with cancer cell, such as, Panc-1 cells, orbeing delivered to a patient.

Gn used in this example was found to have 1-3 layers and was <10 mm (x-ydimension) in size. The Gn used was further functionalized withcarboxylic acid groups to form fGn. After sonication andfunctionalization, the size of the graphene decreased to <300-500 nm(x-y dimension). The surface functionalities enhanced the stability andsolubility of the graphene sheets by generating negative charges on thesurface with a zeta potential of 47.6 mV.

In the example, PTL was loaded onto the surface of fGn throughhydrophobic interactions. To determine the amount of PTL and DMAPT boundto fGn, UV-Vis spectra of drug-free fGn and PTL-fGn were recorded. Thesuccessful attachment of PTL onto the fGn surface was evidenced by thecharacteristic absorbance peak measured at 207 nm, and the concentrationof PTL was determined based on the intensity of this UV absorption band.A loading efficiency of 98.7% was calculated, indicating that almost allof the PTL drug molecules were attached to the surface of fGn. In oneembodiment, the hydrophobic structure of PTL causes it to interact withthe carbon surface of the nanomaterial and that the carboxylate groupsof fGn allow the PTL-fGn complexes to disperse readily in water. In oneembodiment, the drug binds to the nanomaterial via physical absorption(p-p stacking).

The lipophilic sesquiterpene lactone, PTL, has been shown to havepromise as an anticancer agent for the treatment of both solid andhematological tumors. However, its poor solubility poses an importantchallenge to the clinical efficacy and widespread usage of this drug. Tosolve the problem of poor bioavailability and cellular delivery, in thisexample, a carbon-based 2D nanomaterial, fGn, is used to enhance thecellular uptake and increase the solubility of the drug. It is shown inthis example that PTL can be loaded onto fGn nanosheets and that Panc-1cells exposed to this PTL-fGn complex show increased cytotoxicity ascompared to PTL, alone, as indicated by markedly lower IC50 values,increased ROS levels, increased MMP disruption, more active caspase, andincreased staining by nuclear dyes indicative of apoptosis.

In certain embodiments, the fGn increases the efficacy of PTL byassisting in the drug-internalization process through two differentmechanisms: (i) fGn provides a highly reactive surface for the drugadsorption based on various chemical interactions, therefore enhancingthe water-solubility of the drug and the final complexes; and (ii) fGnimproves cellular drug uptake into cells, most probably throughendocytosis, as carried by the graphene sheets into cells, based onrelatively intense electrostatic and hydrophobic interactions. Both ofthese internalization processes likely produce a significant increase inintracellular drug concentrations, resulting in increased cellularcytotoxicity at reduced drug concentrations. Therefore, optimization ofthe chemical surface functionalization of the graphene structures couldbe the foundation of a technologically tunable approach by whichhydrophobic drugs can be solubilized and better delivered to the cells.

Example 7 Attachment of Combined Calcium-Channel Blocker and Anti-CancerDrug to Iron Oxide Nanoparticles for Reducing Chemoresistance of CancerCells

Multidrug resistance (MDR) is one of the major reasons for the failureof chemotherapy in curing advanced cancers. In this example, both theanticancer drug doxorubicin (DOX) and the drug resistance inhibitorverapamil are attached to iron oxide nanoparticles for combined therapyof cancer cells. The description of the nanocomposite can be found inMahmood et al, Therapeutic delivery, 2014, 5(7): 763-780, which isincorporated herein in its entirety by reference.

Iron oxide nanoparticles were purchased from Ocean Nanotech (AR, USA)with an average diameter of 25 nm, COO— groups on the surface, andamphiphilic polymer coating for stability. DOX hydrochloride andverapamil were purchased from Sigma-Aldrich (MO, USA). Thedrug-sensitive breast cancer cell line (MCF-7WT) was purchased fromAmerican Type Culture Collection (ATCC, VA, USA) and maintained in DMEMmedium supplemented by 10% fetal bovine serum (FBS), 100 U/ml penicillinand 100 U/ml streptomycin at 37° C. under 5% CO₂ while thedrug-resistant cancer cell line (NCI/ADRRES) was kindly provided by theNational Cancer Institute and incubated in RPMI-1640 medium containing10% (FBS), 100 U/ml penicillin and 100 U/ml streptomycin and incubatedunder the same conditions. The cells were sub-cultured when confluentwith 0.25% trypsin-EDTA (Gibco BRL, MD, USA).

Drug loading was first processed by mixing the nanoparticles with thedrug in different ratios: basically, for each 10 μg/ml of iron oxidenanoparticles (IONPs), increasing concentrations of 0.5, 0.75 and 1 μgof DOX were added in separate tubes to be described as (DOX-0.5,DOX-0.75 and DOX-1) for DOX alone, (DOX-0.5IO, DOX-0.75IO and DOX-1IO)for DOX with IONPs. Samples were first processed by sonication for 1 hto obtain a uniformly dispersed solution; they were next mixed with DOXsolution and vortexed for at least 4 h at room temperature. A magneticseparator was then applied to the final solution to remove the freedrug. The nanoparticles were washed with distilled water and separatedfor an additional three times. The initial DOX and free DOX contentafter separation were quantified using UV-Vis absorbance with a main DOXpeak appearing at 485 nm using a Shimadzu UV 3600 spectrophotometer(Kyoto, Japan). We calculated the free drug concentration in thesupernatant of the DOX-1IO sample after separation, using a DOXextinction coefficient of 11,000 l/mol/cm at 485 nm. The amount of drugloading efficiency on the IONPs was calculated by using the followingequation: (1−free DOX/initial DOX)×100%. The loading efficiency wascalculated for DOX-1IO and assumed to be unchanged for the otherconcentrations used in this example.

In one embodiment, the IONP core is coated with an oleic acid layer thatis lipophilic and another layer of amphiphilic polymer. The hydrophobicDOX molecules are expected to incorporate into the polymer and be wellattached on the oleic acid layer. The loading amount of DOX onto theIONPs' surface was revealed in the UV-Vis absorption spectra, in whichthe characteristic peaks at 485 nm for DOX were identified. The resultsdemonstrated that DOX efficiently loaded onto the IONPs. The loadingcapacity was found to be about 0.48 μg of DOX when 1 μg of DOX wasloaded for each 10 μg of IONPs. No aggregation phenomenon was observed,which means that the NP-drug complex solution remained highly stable.Since the electrostatic interaction between the positive drug DOX andnegative IONPs may change zeta potential and probably result inaggregation, the loading amount of the drug is very important. We used arelatively low weight percentage of DOX-loading capacity so that the NPsremained in a high enough electrostatic repulsion to preventaggregation.

The drug attachment to the nanoparticles' surface was verified byvarious methods. Transmission electron microscopy (TEM) showed thepresence of a thin layer of the drug molecules loaded on thenanoparticles' surface. The mean particle diameter for IONPS and DOX-1IOwas determined by TEM. The majority of IONPs had an average diameter of26.5±2.1 nm, whereas the DOX-1IO had an average of 32.1±3.1. Inaddition, the interaction between the drug doxorubicin and the ironnanoparticles was determined by XPS, for example, identified based onpeak shift of certain peaks. These results indicate the presence of theDOX layer on the surface of the nanoparticles. Supermagnetic IONPs wereused because their magnetic properties could be implicated formultimodal cancer treatment by using destructive hyperthermia inaddition to our combinatorial therapy.

This example studied the reversal of drug resistance in MCF-7WT andNCI/ADRRES cells using 25 nm IONPs as the carrier for the anticancerdrug doxorubicin. IO with about 50% drug DOX loading efficiency wasdelivered in combination with verapamil to overcome multidrugresistance. The overall results suggest that the use of IONPs as acarrier of commonly used chemotherapeutic drugs represents an excellentchoice for the treatment of various drug-resistant cancers. In addition,in certain embodiments, this approach may be further improved bymodifying the nanoparticles' surface for better drug loading efficiencyand by using a third generation of the calcium-channel blockers toovercome the resistance.

The foregoing description of the exemplary embodiments of the inventionhas been presented only for the purposes of illustration and descriptionand is not intended to be exhaustive or to limit the invention to theprecise forms disclosed. Many modifications and variations are possiblein light of the above teaching.

The embodiments were chosen and described in order to explain theprinciples of the invention and their practical application so as toenable others skilled in the art to utilize the invention and variousembodiments and with various modifications as are suited to theparticular use contemplated. Alternative embodiments will becomeapparent to those skilled in the art to which the invention pertainswithout departing from its spirit and scope. Accordingly, the scope ofthe invention is defined by the appended claims as well as the inventionincluding drawings.

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What is claimed is:
 1. A nanoagent comprising at least onenanocomposite, the nanocomposite comprising: at least one gold nanorod;a silver layer coated on an outer surface of the gold nanorod, thesilver layer comprising silver nanoparticles; a Raman reporter moleculelayer coated on the silver layer, wherein the Raman reporter moleculelayer comprises the Raman reporter molecules that are detectable bysurface enhanced Raman spectroscopy (SERS); a pegylated layer coated onthe Raman reporter molecule layer, comprising at least one of thiolatedpolyethylene glycol (HS-PEG), thiolated polyethylene glycol acid(HS-PEG-COOH) and HS-PEG-NHx; and an active layer conjugated to thepegylated layer, the active layer comprising at least one of: atargeting molecule configured to bind to a target of interest; and afunctional molecule configured to interact with the target of interest.2. The nanoagent of claim 1, wherein the target of interest is at leastone of a cancer cell, a pathogen, or a plant.
 3. The nanoagent of claim1, wherein the targeting molecule comprises an antibody specificallybinds to the target of interest.
 4. The nanoagent of claim 1, whereinthe functional molecule is a virus, a phage, a drug, a growth factor,antibiotics, a gene, a plasmid, a vaccine, a plant growth agent, ananti-fungal, a fertilizer, herbicides, an antibody that specificallybinds to S100 calcium-binding protein B (S100B), or other biologicalactive molecules.
 5. The nanoagent of claim 1, wherein the active layercomprises a biomarker of traumatic brain injury (TBI) or an antibodythat specifically binds to the biomarker.
 6. The nanoagent of claim 5,wherein the biomarker of TBI comprises at least one of S100calcium-binding protein B (S100B), neuron-specific enolase (NSE), myelinbasic protein (MBP), caspase-3, interleukins, tau protein, neurofilamentlight polypeptide (NEFL), neurofilament heavy polypeptide (NEFH), glialfibrillary acidic protein, amyloid precursor protein (APP), and amyloid.7. The nanoagent of claim 1, wherein the gold nanorod has an aspectratio (AR) in a range of about 1-9, a length in a range of about 10-100nm, and a diameter in a range of about 1-40 nm; and wherein the silverlayer has a thickness in a range of about 0.5-5 nm.
 8. The nanoagent ofclaim 1, wherein the Raman reporter molecule layer comprises4-mercaptobenzoic acid (4MBA), p-aminothiophenol (PATP),p-nitrothiophenol (PNTP), 4-(methylsulfanyl)thiophenol (4MSTP), or othermolecules with unique Raman spectra and intense Raman peak intensities.9. The nanoagent of claim 1, wherein the HS-PEG has a molecular weightin a range of about 1.5-15 kilo Dalton (kD) and the HS-PEG-COOH has amolecular weight in a range of about 1-10 kD.
 10. The nanoagent of claim1, wherein at least one of the targeting molecule and the functionalmolecule is conjugated to the pegylated layer through the carboxylicgroup of the HS-PEG-COOH or amine group of the HS-PEG-NHx.
 11. Thenanoagent of claim 1, wherein the targeting molecule comprisesanti-epithelial cell adhesion molecule antibody (anti-EpCAM), anti-CD44antibody, anti-insulin-like growth factor 1 receptor antibody(anti-IGF-1), anti-Keratin 18 antibody, or one or more antibodiesspecific to the target of interest.
 12. The nanoagent of claim 11,wherein the at least one nanocomposite comprises a first nanocomposite,a second nanocomposite, a third nanocomposite, and a fourthnanocomposite; wherein the Raman reporter molecule layer of the firstnanocomposite comprises 4-mercaptobenzoic acid (4MBA), and the antibodyof the first nanocomposite is anti-epithelial cell adhesion moleculeantibody (anti-EpCAM); wherein the Raman reporter molecule layer of thesecond nanocomposite comprises p-aminothiophenol (PATP), and theantibody of the second nanocomposite is anti-CD44 antibody; wherein theRaman reporter molecule layer of the third nanocomposite comprisesp-nitrothiophenol (PNTP), and the antibody of the third nanocomposite isanti-insulin-like growth factor 1 receptor antibody (anti-IGF-1);wherein the Raman reporter molecule layer of the fourth nanocompositecomprises 4-(methylsulfanyl)thiophenol (4MSTP), and the antibody of thefourth nanocomposite is anti-Keratin 18 antibody; and wherein SERSsignal corresponding to each of the first, second, third and fourthnanocomposites is represented by a predetermined color.
 13. A system fordetecting a target of interest and monitoring conditions of the targetof interest, comprising: a nanoagent of claim 1; a surface enhancedRaman spectrometer configured to provide an incident radiation signal tothe target of interest, and to collect SERS signals generated by theRaman reporter molecule layer in response to the incident radiationsignal; and a processing unit for processing the SERS signals collectedby the surface enhanced Raman spectrometer, so as to detect and monitorthe conditions of the target of interest.
 14. A method of making ananocomposite, comprising: forming at least one gold nanorod; coating asilver layer on an outer surface of the gold nanorod; assembling a Ramanreporter molecule layer on the coated silver layer; coating a pegylatedlayer on the assembled Raman reporter molecule layer; conjugating thecoated pegylated layer with an active layer, the active layer comprisingat least one of a targeting molecule configured to bind to the target ofinterest and a functional molecule configured to interact with thetarget of interest.
 15. The method of claim 14, wherein the step offorming the at least one gold nanorod comprises: mixing a firstexadecyltrimethylammoniumbromide (CTAB) solution with a silver nitratesolution to form a first mixture; adding a first HAuCl₄ to the firstmixture to form a second mixture; adding a first ascorbic acid to thesecond mixture to form a third mixture; adding a seed solution to thethird mixture to form a fourth mixture; and centrifuging the fourthmixture to form a first precipitate, wherein the first precipitatecomprises the gold nanorod.
 16. The method of claim 15, wherein the seedsolution is prepared by: mixing a second CTAB solution with a secondHAuCl₄ to form a fifth mixture; and adding NaBH₄ to the fifth mixtureand stirring to form the seed solution.
 17. The method of claim 16,wherein the step of coating the silver layer comprises: dispersing thegold nanorod in a third CTAB solution by sonication to form a sixthmixture; adding a polyvinylpyrrolidone (PVP) solution and AgNO₃ to thesixth mixture and gently mixing to form a seventh mixture; adding asecond ascorbic acid to the seventh mixture to form an eighth mixture;adding NaOH solution to the eighth mixture to form a ninth mixture, suchthat the pH of the ninth mixture is elevated to about pH 9, and a silverion reduction reaction is initiated; and centrifuging the ninth mixtureto form a second precipitate, wherein the second precipitate comprisesthe gold nanorod coated with the silver layer.
 18. The method of claim17, wherein the step of assembling the Raman reporter molecule layercomprises: dispersing the gold nanorod coated with the silver layer indistilled water to form a tenth mixture; dissolving the Raman reportermolecule comprising 4-mercaptobenzoic acid (4-MBA), p-aminothiophenol(PATP), p-nitrothiophenol (PNTP), or 4-(methylsulfanyl)thiophenol(4-MSTP), in ethanol to form a reporter solution; adding the reportersolution to the tenth mixture and stirring to form an eleventh mixture;and centrifuging the eleventh mixture to form a third precipitate,wherein the third precipitate comprises the gold nanorod coated with thesilver layer, and assembled with the Raman report molecule layer. 19.The method of claim 18, wherein the step of coating the pegylated layercomprises: dispersing the gold nanorod with the coated silver layer andthe assembled Raman reporter molecule layer in thiolated polyethyleneglycol acid (HS-PEG-COOH) solution and vigorously stirring to form atwelfth mixture, wherein the HS-PEG-COOH solution comprises about 2mg/ml HS-PEG and about 2 mM NaCl; adding thiolated polyethylene glycol(HS-PEG) to the twelfth mixture and keep at about 5° C. overnight toform a thirteenth mixture; and centrifuging the thirteenth mixture toform a fourth precipitate, wherein the fourth precipitate comprises thegold nanorod coated with the silver layer, assembled with the Ramanreport molecule layer, and coated with the pegylated layer.
 20. Themethod of claim 19, wherein the step of conjugating the coated pegylatedlayer with the targeting molecule comprises: suspending the gold nanorodwith the coated silver layer, the assembled Raman reporter moleculelayer, and the coated with pegylated layer in phosphate-buffered saline(PBS) by sonicating to form a suspending mixture; addingN-hydroxysuccinimide (NHS) and 1N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to the suspending mixture and stirringto form a fourteenth mixture; washing the fourteenth mixture bycentrifuging twice using PBS to obtain a fifth precipitate; dispendingthe fifth precipitate in PBS to form a fifteen mixture; adding moleculesof the targeting molecule to the fifteenth mixture and mixing thoroughlyto form a sixteen mixture, wherein the targeting molecule comprisesanti-EpCAM, anti-CD44, anti-IGF-1 Receptor β, anti-Keratin 18, or one ormore antibodies specific to the target of interest; and stirring thesixteenth mixture at room temperature to form the nanocomposite.
 21. Ananocomposite, comprising: a nanostructure formed by at least onenanomaterial; and an active layer conjugated to the nanostructure, andcomprising at least one of: a targeting molecule configured to bind to atarget of interest; and a functional molecule configured to interactwith a target of interest.
 22. The nanocomposite of claim 21, whereinthe nanostructure has a spherical shape, a tubular shape, a cylindricalshape, or a rod-like triangular shape.
 23. The nanocomposite of claim21, wherein the nanomaterial comprises at least one of silver coatedgold rods, quantum dots, nanowires, nanotubes, nanofibers, andfullerenes.
 24. The nanocomposite of claim 21, wherein the nanomaterialcomprises gold, silver, gold/silver, copper, iron, Fe_(x)O_(y), TiO₂,SiO₂, and carbon.
 25. The nanocomposite of claim 21, wherein the targetof interest is at least one of a cancer cell, a pathogen, or a plant,and the functional molecule is a virus, a phage, a drug, a growthfactor, antibiotics, a gene, a plasmid, a vaccine, a plant growth agent,an anti-fungal, a fertilizer, herbicides, an antibody that specificallybinds to S100 calcium-binding protein B (S100B), or other biologicalactive molecules.
 26. The nanocomposite of claim 25, wherein the targetof interest is the cancer cell, and the functional molecule is the viruscapable of reproducing in the cancer cell.
 27. The nanocomposite ofclaim 25, wherein the target of interest is the plant, and thefunctional molecule is the growth factor capable of promoting growth ofthe plant.
 28. The nanocomposite of claim 21, wherein the active layercomprises a biomarker of traumatic brain injury (TBI) or an antibodythat specifically binds to the biomarker.
 29. The nanocomposite of claim28, wherein the biomarker of TBI comprises at least one of S100calcium-binding protein B (S100B), neuron-specific enolase (NSE), myelinbasic protein (MBP) caspase-3, interleukins, tau protein, neurofilamentlight polypeptide (NEFL), neurofilament heavy polypeptide (NEFH), glialfibrillary acidic protein, amyloid precursor protein (APP), and amyloid.30. The nanocomposite of claim 21, wherein the nanostructure comprises:at least one gold nanorod; and a silver layer surrounding the at leastone gold nanorod, the silver layer comprising silver nanoparticles. 31.The nanocomposite of claim 30, wherein the gold nanorod has an aspectratio (AR) in a range of about 1-9, a length in a range of about 10-100nm, and a diameter in a range of about 1-40 nm; and wherein the silverlayer has a thickness in a range of about 0.5-5 nm.
 32. Thenanocomposite of claim 21, further comprising a reporter layer disposedbetween the nanomaterial and the active layer, wherein the reporterlayer is detectable by at least one of surface enhanced Ramanspectroscopy (SERS), magnetic resonance imaging (MRI), x-rayradiography, computed tomography (CT), and infrared spectroscopy (IR).33. The nanocomposite of claim 32, wherein the reporter layer comprises4-mercaptobenzoic acid (4MBA), p-aminothiophenol (PATP),p-nitrothiophenol (PNTP), 4-(methylsulfanyl)thiophenol (4MSTP), or othermolecules with unique Raman spectra and intense Raman peak intensities.34. The nanocomposite of claim 32, further comprising a pegylated layerdisposed between the reporter layer and the active layer, wherein thepegylated layer comprises at least one of thiolated polyethylene glycol(HS-PEG), thiolated polyethylene glycol acid (HS-PEG-COOH) andHS-PEG-NHx.
 35. The nanocomposite of claim 21, wherein the targetingmolecule is molecule of anti-epithelial cell adhesion molecule antibody(anti-EpCAM), anti-CD44 antibody, anti-insulin-like growth factor 1receptor antibody (anti-IGF-1), anti-Keratin 18 antibody, or one or moreantibodies specific to the target of interest.
 36. The nanocomposite ofclaim 35, wherein the functional molecule is conjugated to at least oneof the pegylated layer and the targeting molecule.